Xanthene dyes

ABSTRACT

The invention provides a novel class of xanthene dyes, some of which are functionalized to allow their coupling to conjugation partners of interest, e.g. biomolecules, drugs, toxins and the like. Also provided are conjugates of the dyes, methods of preparing and using the dyes and their conjugates and kits including the dyes and their conjugates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.12/036,926, filed on Feb. 25, 2008; which is a Continuation of U.S.patent application Ser. No. 10/824,175, filed on Apr. 13, 2004, now U.S.Pat. No. 7,344,701, which claims priority under 35 U.S.C. §119(e) toU.S. Provisional Patent Application No. 60/541,686, filed on Feb. 3,2004, the disclosures of which are incorporated by reference herein intheir entirety for all purposes.

FIELD OF INVENTION

The present invention relates generally to the synthesis of fluorescentcompounds that are analogues of xanthene dyes. The compounds of theinvention are fluorophores that are derivatized to allow their facileattachment to a carrier molecule.

BACKGROUND

There is a continuous and expanding need for rapid, highly specificmethods of detecting and quantifying chemical, biochemical andbiological substances as analytes in research and diagnostic mixtures.Of particular value are methods for measuring small quantities ofnucleic acids, peptides, saccharides, pharmaceuticals, metabolites,microorganisms and other materials of diagnostic value. Examples of suchmaterials include narcotics and poisons, drugs administered fortherapeutic purposes, hormones, pathogenic microorganisms and viruses,peptides, e.g., antibodies and enzymes, and nucleic acids, particularlythose implicated in disease states.

The presence of a particular analyte can often be determined by bindingmethods that exploit the high degree of specificity that characterizesmany biochemical and biological systems. Frequently used methods arebased on, for example, antigen-antibody systems, nucleic acidhybridization techniques and protein-ligand systems. In these methods,the existence of a complex of diagnostic value is typically indicated bythe presence or absence of an observable “label” which is attached toone or more of the interacting materials. The specific labeling methodchosen often dictates the usefulness and versatility of a particularsystem for detecting an analyte of interest. Preferred labels areinexpensive, safe, and capable of being attached efficiently to a widevariety of chemical, biochemical, and biological materials withoutsignificantly altering the important binding characteristics of thosematerials. The label should give a highly characteristic signal, andshould be rarely, and preferably never, found in nature. The labelshould be stable and detectable in aqueous systems over periods of timeranging up to months. Detection of the label is preferably rapid,sensitive, and reproducible without the need for expensive, specializedfacilities or the need for special precautions to protect personnel.Quantification of the label is preferably relatively independent ofvariables such as temperature and the composition of the mixture to beassayed.

A wide variety of labels have been developed, each with particularadvantages and disadvantages. For example, radioactive labels are quiteversatile, and can be detected at very low concentrations, such labelsare, however, expensive, hazardous, and their use requires sophisticatedequipment and trained personnel. Thus, there is wide interest innon-radioactive labels, particularly in labels that are observable byspectrophotometric, spin resonance and luminescence techniques, andreactive materials, such as enzymes that produce such molecules.

Labels that are detectable using fluorescence spectroscopy are ofparticular interest, because of the large number of such labels that areknown in the art. Moreover, as discussed below, the literature isreplete with syntheses of fluorescent labels that are derivatized toallow their attachment to other molecules and many such fluorescentlabels are commercially available.

In addition to being directly detected, many fluorescent labels operateto quench the fluorescence of an adjacent second fluorescent label.Because of its dependence on the distance and the magnitude of theinteraction between the quencher and the fluorophore, the quenching of afluorescent species provides a sensitive probe of molecular conformationand binding, as well as or other interactions. An excellent example ofthe use of fluorescent reporter quencher pairs is found in the detectionand analysis of nucleic acids.

Fluorescent nucleic acid probes are important tools for geneticanalysis, in both genomic research and development, and in clinicalmedicine. As information from the Human Genome Project accumulates, thelevel of genetic interrogation mediated by fluorescent probes willexpand enormously. One particularly useful class of fluorescent probesincludes self-quenching probes, also known as fluorescence energytransfer probes, or FET probes. The design of different probes usingthis motif may vary in detail. In an exemplary FET probe, both afluorophore and a quencher are tethered to a nucleic acid. The probe isconfigured such that the fluorophore is proximate to the quencher andthe probe produces a signal only as a result of its hybridization to anintended target. Despite the limited availability of FET probes,techniques incorporating their use are rapidly displacing alternativemethods.

Probes containing a fluorophore-quencher pair have been developed fornucleic acid hybridization assays where the probe forms a hairpinstructure, i.e., where the probe hybridizes to itself to form a loopsuch that the quencher molecule is brought into proximity with thereporter molecule in the absence of a complementary nucleic acidsequence to prevent the formation of the hairpin structure (see, forexample, WO 90/03446; European Patent Application No. 0 601 889 A2).When a complementary target sequence is present, hybridization of theprobe to the complementary target sequence disrupts the hairpinstructure and causes the probe to adopt a conformation where thequencher molecule is no longer close enough to the reporter molecule toquench the reporter molecule. As a result, the probes provide anincreased fluorescence signal when hybridized to a target sequence thanwhen they are unhybridized.

Assays have also been developed for detecting a selected nucleic acidsequence and for identifying the presence of a hairpin structure usingtwo separate probes, one containing a reporter molecule and the other aquencher molecule (see, Meringue, et al., Nucleic Acids Research, 22:920-928 (1994)). In these assays, the fluorescence signal of thereporter molecule decreases when hybridized to the target sequence dueto the quencher molecule being brought into proximity with the reportermolecule.

One particularly important application for probes including areporter—quencher molecule pair is their use in nucleic acidamplification reactions, such as polymerase chain reactions (PCR), todetect the presence and amplification of a target nucleic acid sequence.In general, nucleic acid amplification techniques have opened broad newapproaches to genetic testing and DNA analysis (see, for example,Arnheim et al. Ann. Rev. Biochem., 61: 131-156 (1992)). PCR, inparticular, has become a research tool of major importance withapplications in, for example, cloning, analysis of genetic expression,DNA sequencing, genetic mapping and drug discovery (see, Arnheim et al.,supra; Gilliland et al., Proc. Natl. Acad. Sci. USA, 87: 2725-2729(1990); Bevan et al., PCR Methods and Applications, 1: 222-228 (1992);Green et al., PCR Methods and Applications, 1: 77-90 (1991); Blackwellet al., Science, 250: 1104-1110 (1990)).

Commonly used methods for detecting nucleic acid amplification productsrequire that the amplified product be separated from unreacted primers.This is typically achieved either through the use of gelelectrophoresis, which separates the amplification product from theprimers on the basis of a size differential, or through theimmobilization of the product, allowing free primer to be washed away.However, a number of methods for monitoring the amplification processwithout prior separation of primer have been described; all of them arebased on FET, and none of them detect the amplified product directly.Instead, the methods detect some event related to amplification. Forthat reason, they are accompanied by problems of high background, andare not quantitative, as discussed below.

One method, described in Wang et al. (U.S. Pat. No. 5,348,853; and Anal.Chem., 67: 1197-1203 (1995)), uses an energy transfer system in whichenergy transfer occurs between two fluorophores on the probe. In thismethod, detection of the amplified molecule takes place in theamplification reaction vessel, without the need for a separation step.

A second method for detecting an amplification product without priorseparation of primer and product is the 5′-nuclease PCR assay (alsoreferred to as the TaqMan™ assay) (Holland et al., Proc. Natl. Acad.Sci. USA, 88: 7276-7280 (1991); Lee et al., Nucleic Acids Res., 21:3761-3766 (1993)). This assay detects the accumulation of a specific PCRproduct by hybridization and cleavage of a doubly labeled fluorogenicprobe (the “TaqMan” probe) during the amplification reaction. Thefluorogenic probe consists of a nucleic acid labeled with both afluorescent reporter dye and a quencher dye. During PCR, this probe iscleaved by the 5′-exonuclease activity of DNA polymerase if, and onlyif, it hybridizes to the segment being amplified. Cleavage of the probegenerates an increase in the fluorescence intensity of the reporter dye.

Yet another method of detecting amplification products that relies onthe use of energy transfer is the “beacon probe” method described byTyagi et al. (Nature Biotech., 14: 303-309 (1996)) which is also thesubject of U.S. Pat. No. 5,312,728 to Lizardi et al. This method employsnucleic acid hybridization probes that can form hairpin structures. Onone end of the hybridization probe (either the 5′- or 3′-end) there is adonor fluorophore, and on the other end, an acceptor moiety. In thismethod, the acceptor moiety is a quencher, absorbing energy from thedonor. Thus when the beacon is in the open conformation, thefluorescence of the donor fluorophore is detectable, whereas when thebeacon is in hairpin (closed) conformation, the fluorescence of thedonor fluorophore is quenched. When employed in PCR, the molecularbeacon probe, which hybridizes to one of the strands of the PCR product,is in “open conformation,” and fluorescence is detected, while thosethat remain unhybridized will not fluoresce. As a result, the amount offluorescence will increase as the amount of PCR product increases, andthus can be used as a measure of the progress of the PCR.

The probes discussed above are most generally configured such that thequencher and fluorophore are on the 3′- and 5′-ends of the probe(Lyamichev et al., Science, 260:778-783 (1993)). This spacing of thefluorophore and quencher may impede fluorescent energy transfer:fluorescence energy transfer decreases as the inverse sixth power of thedistance between the fluorophore and quencher. Thus, if the quencher isnot close enough to the reporter to achieve efficient quenching thebackground emissions from the probe can be quite high.

For the xanthene dye to be useful as a label it must posses a chemicalfunctional group the will permit it to bind to, or react with, asubstrate of interest. The incorporation of such reactive chemicalfunctionality into xanthene dyes usually requires additional syntheticsteps and/or difficult to implement purification methods. In particular,separation of structural isomers of fluoresceins and rhodamines, whichare the most commonly used xanthene labeling reagents for biological andmedical applications, is tedious and is to be avoided if possible.

The core chemical structure of many fluorescein and rhodamine dyesincludes a carboxylic acid group in the ortho position of the benzenering attached to the xanthene residue; some others posses a sulfonicacid group at this site. The carboxylic acid group has not been widelyutilized as a site for conjugation of the dye to substrates due to itslow reactivity and due to side reactions that render the dyenon-fluorescent. Although the carboxylic acid can be activated andreacted with alcohols to form esters or with amines to form amides, theester linkage is of insufficient stability to be useful when preparingcompounds that are stably labeled with a fluorophore.

The amide linkage is stable to hydrolysis but while some amides preparedfrom the activated carboxylic acid and primary amines are reported to becolored (Mayer et al., U.S. Pat. No. 4,647,675) others are reported toundergo a spirolactamization reaction in which the dye loses its colorand is rendered non-fluorescent (Adamczyk et al., Synthetic Commun. 31:2681-2690 (2001); and Cincotta et al., U.S. Pat. No. 4,290,955)). Incontrast, secondary amines react with the activated carboxylic acid tocreate an amide link that cannot undergo spirolactamization, providing axanthene dye that retains its color and fluorescence (Gao et al., WO02/055512). Menchen et al. disclose xanthene dyes in which the orthocarboxyl moiety is activated and coupled to another species. Other amidederivatized xanthene dyes are disclosed in Haugland et al., U.S. Pat.No. 6,399,392; and Mayer et al., U.S. Pat. No. 4,935,059.

Xanthene dyes that posses a sulfonic acid group in the ortho positioncan be activated and reacted with alcohols and with amines in a mannersimilar to xanthene dyes with ortho carboxylic acid groups to yieldsulfonate esters and sulfonamides, respectively. The sulfonate estersare not stable under aqueous conditions and are of little use as linkerfunctionality for preparing oligonucleotides. The sulfonamides arestable and have been used to prepare reactive xanthene dyes such assuccinimidyl esters, maleimides and phosphoramidites.

None of the above-described references discloses or suggests themodification of the fluorophore nucleus with a versatile amide-linkedmoiety that allows for the facile variation of the composition, lengthand degree of branching of the linker. Furthermore, none of thereferences suggest a linker that provides a locus for attaching thefluorophore to a solid support, nor do the references describe abranched linker moiety that tethers both a phosphoramidite and adimethoxytrityl ether to a single endocyclic nitrogen atom.

Attaching quenchers or fluorophores to sites other than the readilyaccessible 5′-OH group generally requires the synthesis of fluorescentlabels that are of use to attach the fluorophore to a single reactiveresidue of a carrier molecule or a selected reactive functional group onthat residue; reacting the same fluorophore with a different functionalgroup of the carrier generally requires a new modification of thefluorescent core. Similarly, modifying the structure or composition ofthe linker arm requires a modification to the fluorophore nucleus. Thus,a xanthene label that provides a versatile entry point for an array ofsynthetic modifications would represent a significant advance in theart.

BRIEF SUMMARY OF THE INVENTION

The inventors have prepared a class of xanthene-based fluorophoresmodified with a versatile linker arm, the structure of which is readilyalterable, allowing the conjugation of the label to a variety ofpositions, through diverse functional groups, on a carrier molecule. Thexanthene-based labels are readily attached to a carrier molecule usingtechniques well known in the art, or modifications of such techniquesthat are well within the abilities of those of skill in the art. Theversatility of the labels set forth herein provides a marked advantageover currently utilized xanthene labels, probes assembled using theselabels and methods relying upon such labels and probes. Moreover, thepresent invention provides a class of chemically versatile labels inwhich the fluorophore can be engineered to have a desired light emissionprofile.

In an exemplary embodiment, the fluorescent nucleus is functionalizedthrough the ortho carboxylic acid group of the phenyl moiety attached tothe xanthene nucleus, requiring fewer chemical synthesis steps andeliminating the need to separate structural isomers. Additionally, manyxanthene dyes possessing the ortho carboxy functional group arecommercially available at low cost for use as starting materials.

Thus, in a first aspect, the present invention provides a fluorescentcompound having the formula:

in which R¹, R² and R⁴-R¹¹ are each independently selected from groupssuch as substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,halogen, H, NO₂, CN and C(Z¹)R¹⁴, NR¹⁵R¹⁶ and Z²R¹⁶. R³ is selected fromZ²R¹⁶ and NR¹⁵R¹⁶.

Z¹ represents O, S or NH. Z² is either O or S. Groups corresponding toR¹⁵ include H, substituted or unsubstituted alkyl, and substituted orunsubstituted heteroalkyl. R¹⁶ is selected from H, substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl andC(Z³)R¹⁷. R¹⁵ and R¹⁶, together with the nitrogen to which they areattached, can also be any nitrogen-containing reactive group. Exemplarygroups include —NHNH₂, —N═C═S and —N═C═O.

Z³ represents O, S or NH. The symbol R¹⁷ represents groups such assubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, OR¹⁸, and NR¹⁹R²⁰. R¹⁸ represents H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and C(O)R²¹. R¹⁹ and R²⁰ are symbols representing groupsindependently selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl. R²¹ is substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl.

The symbol Y represents either C(O) or S(O)₂. X is (NR²²R²³) or (O). R²²and R²³ are independently selected and are members selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl.

R¹² or R¹³ are independently selected from substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl, with the proviso that at leastone of R¹² or R¹³ comprises an oxygen-containing reactive group acarrier molecule or solid support to which the dye of the invention isconjugated through a moiety produced by reaction of theoxygen-containing reactive group with a reactive group on the carriermolecule of complementary reactivity. R¹² and R¹³, together with thenitrogen atom to which they are attached are optionally joined to form aring. Exemplary rings include 4-6-membered heterocylic and heteroarylrings. When R¹² and R¹³, together with the nitrogen to which they areattached form a piperazine ring in which the second nitrogen atom isfunctionalized with an alkyl moiety bearing an oxygen-containingreactive group, the reactive group is preferably a phosphoramidite.

The bond formed between through the reactive oxygen-containingfunctional group and the group of complementary reactivity is, forexample, a bond to a carrier molecule, a bond to a linker bound to acarrier molecule, a bond to a solid support, a bond to a linker bound toa solid support, a bond to a fluorescence quencher, and a bond to alinker bound to a fluorescence quencher.

The substituents on the aryl ring nuclei can be joined to form rings.For example, in one embodiment in which R³ is NR¹⁵R¹⁶, R², R⁴ and R¹⁵and R¹⁶, together with the nitrogen atom to which they are bound, arefused with the phenyl moiety to which NR¹⁵R¹⁶, R² and R⁴ are bound,forming a substituted or unsubstituted ring system having the generalformula:

In yet another embodiment in which X is NR²²R²³, R⁵, R⁶ and R²² and R²³,together with the nitrogen atom to which they are bound, are fused withthe unsaturated 6-member ring to which NR²²R²³, R⁵ and R⁶ are bound,forming a substituted or unsubstituted ring system having the generalformula:

The present invention also provides a conjugate between a carriermolecule, e.g., a nucleic acid, and a fluorescent compound of theinvention, which is covalently or ionically bound to a moiety of thecarrier molecule. When the carrier molecule is a nucleic acid,representative moieties at which the fluorescent compounds of theinvention are attached include the sugar moiety, at O- and/or C-centers;endo- and/or exo-cyclic amines, carbon atoms of nucleobase moieties, andinternucleotide bridges. In still a further exemplary embodiment, theconjugate between the compound of the invention and the carrier moleculeincludes at least one moiety that quenches the fluorescence emission ofthe compound of the invention.

Also provided are assays utilizing one or more compound of the inventionor a conjugate between a compound of the invention and a carriermolecule. In exemplary assays the carrier molecule includes both a dyeof the invention and a species that quenches fluorescence emission fromthe dye.

Other aspects, embodiments and objects of the present invention will beapparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B displays representative compounds of the invention.

FIG. 2 is a representative reverse phase HPLC trace of3′-TTCGATAAGTCTAG-5′, 5′ labeled with 15.

FIG. 3 is a representative mass spectrum of 3′-TTCGATAAGTCTAG-5′, 5′labeled with 15.

FIG. 4 is a representative anion exchange HPLC trace of3′-TTCGATAAGTCTAG-5′, 5′ labeled with 25.

FIG. 5 is a representative mass spectrum of 3′-TTCGATAAGTCTAG-5′, 5′labeled with compound 25.

FIG. 6 is a representative reverse phase HPLC trace of3′-TTCGATAAGTCTAG-5′, 5′ labeled with compound 19.

FIG. 7 is a representative mass spectrum of 3′-TTCGATAAGTCTAG-5′, 5′labeled with compound 19.

FIG. 8 is a representative anion exchange HPLC trace of3′-TTCGATAAGTCTAG-5′, 3′ labeled by using dye bearing CPG compound 29.

FIG. 9 is a representative mass spectrum of 3′-TTCGATAAGTCTAG-5′, 3′labeled by using dye bearing CPG compound 29.

FIG. 10 is an overlay of absorption and emission spectra of5′-TTTTTTTTTT-3′ 5′ labeled with 25.

FIG. 11 is an overlay of absorption and emission spectra of5′-TTTTTTTTTT-3′ 5′ labeled with 15.

FIG. 12 is an overlay of absorption and emission spectra of5′-TTTTTTTTTT-3′ 5′ labeled with 6.

FIG. 13 is an overlay of absorption and emission spectra of5′-TTTTTTTTTT-3′ 5′ labeled with 19.

FIG. 14 is a comparison of the performance of probes labeled with dye 15to probes labeled with JOE. The graph shows the results of the real-timePCR analysis performed on the ABI 7700 Sequence Detection System. Datafor dye 15 and JOE labeled ApoB probes are presented in Ct values. Theresults for the dye 15 labeled probes show that it is at leastequivalent to the JOE labeled probes.

FIG. 15 is a comparison of the performance of probes labeled with dye 15to probes labeled with HEX and JOE. The graph shows the results of thereal-time PCR analysis performed on the ABI 7700 Sequence DetectionSystem. Data for dye 15, HEX and JOE labeled telomerase probes arepresented in Ct values. The results for dye 15 labeled probe show thatit is at least equivalent to the HEX or JOE labeled probes.

FIG. 16 is a comparison of the performance of probes labeled with dye 19to probes labeled with ROX and Texas Red. The graph shows the results ofthe real-time PCR analysis performed on the ABI 7700 Sequence DetectionSystem. Data for dye 19, ROX and Texas Red labeled ApoB probes arepresented in Ct values. The results for the dye 19 labeled probe showthat it is at least equivalent to the ROX or Texas Red labeled probes.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

“FET,” as used herein, refers to “Fluorescence Energy Transfer.”

“FRET,” as used herein, refers to “Fluorescence Resonance EnergyTransfer.” These terms are used herein to refer to both radiative andnon-radiative energy transfer processes. For example, processes in whicha photon is emitted and those involving long-range electron transfer areincluded within these terms. Throughout this specification, both ofthese phenomena are subsumed under the general term “donor-acceptorenergy transfer.”

DEFINITIONS

Where chemical moieties are specified by their conventional chemicalformulae, written from left to right, they equally encompass the moietywhich would result from writing the structure from right to left, e.g.,—CH₂O— is intended to also recite —OCH₂—; —NHS(O)₂— is also intended torepresent. —S(O)₂HN—, etc.

As used herein, “nucleic acid” means any natural or non-naturalnucleoside, or nucleotide and oligomers and polymers thereof, e.g., DNA,RNA, single-stranded, double-stranded, triple-stranded or more highlyaggregated hybridization motifs, and any chemical modifications thereof.Modifications include, but are not limited to, conjugation with acompound of the invention or a construct that includes a compound of theinvention covalently attached to a linker that tethers the compound tothe nucleic acid, and those providing the nucleic acid with a group thatincorporates additional charge, polarizability, hydrogen bonding,electrostatic interaction, fluxionality or functionality to the nucleicacid. Exemplary modifications include the attachment to the nucleicacid, at any position, of one or more hydrophobic or hydrophilicmoieties, minor groove binders, intercalating agents, quenchers,chelating agents, metal chelates, solid supports, and other groups thatare usefully attached to nucleic acids.

Exemplary modified nucleic acids include, but are not limited to,peptide nucleic acids (PNAs), those with phosphodiester groupmodifications (e.g., replacement of O⁻ with OR, NR, or SR), 2′-, 3′- and5′-position sugar modifications, modifications to the base moiety, e.g.,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications, i.e.,substitution of P(O)O₃ with another moiety, methylations, unusualbase-pairing combinations such as the isobases, isocytidine andisoguanidine and the like. Nucleic acids can also include non-naturalbases, e.g., nitroindole. Non-natural bases include bases that aremodified with a compound of the invention or a linker-compound of theinvention construct, a minor groove binder, an intercalating agent, ahybridization enhancer, a chelating agent, a metal chelate, a quencher,a fluorophore, a fluorogenic compound, etc. Modifications within thescope of “nucleic acid” also include 3′ and 5′ modifications with one ormore of the species described above.

“Nucleic acid” also includes species that are modified at one or moreinternucleotide bridge (e.g., P(O)O₃) by replacing or derivatizing anoxygen of the bridge atom with a compound of the invention or a speciesthat includes a compound of the invention attached to a linker. Forexample, “nucleic acid” also refers to species in which the P(O)O₂moiety (the O⁻ moiety remains unchanged or is converted to “OR”) of anatural nucleic acid is replaced with a non-natural linker species,e.g., —ORP(O)O—, —ROP(O)R—, —ORP(O)OR— —ROP(O)OR— or —RP(O)R— in whichthe symbol “-” indicates the position of attachment of the linker to the2′-, 3′- or 5′-carbon of a nucleotide sugar moiety, thus allowing theplacement of the exemplified, and other, non-natural linkers betweenadjacent nucleoside sugar moieties. Exemplary linker subunits (“R”)include substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl moieties. “R” can include a compound of theinvention or a construct of a linker and a compound of the invention.

Furthermore, “nucleic acid” includes those species in which one or moreinternucleotide bridge does not include phosphorus: the bridge beingoptionally modified with a compound of the invention or a linkerarm-xanthene dye construct. An exemplary bridge includes a substitutedor unsubstituted alkyl or substituted or unsubstituted heteroalkylmoiety in which a carbon atom is the locus for the interconnection oftwo nucleoside sugar residues (or linker moieties attached thereto) anda compound of the invention or a linker construct that includes acompound of the invention. The discussion above is not limited tomoieties that include a carbon atom as the point of attachment; thelocus can also be another appropriate linking atom, such as nitrogen oranother atom.

Those of skill in the art will understand that in each of the “nucleicacid” compounds described above, the structure corresponding to the term“compound of the invention” can be interchanged with a quencher, ahybridization enhancer, and intercalator, a minor groove binder, achelating agent, a metal chelate or other moiety that is usefullyconjugated to a nucleic acid, optionally being present in tandem withspecies that include a compound of the invention or a derivativethereof.

As used herein, “quenching group” refers to any fluorescence-modifyinggroup of the invention that can attenuate at least partly the lightemitted by a fluorescent group. This attenuation is referred to hereinas “quenching”. Hence, illumination of the fluorescent group in thepresence of the quenching group leads to an emission signal that is lessintense than expected, or even completely absent. Quenching typicallyoccurs through energy transfer between the fluorescent group and thequenching group.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds, alternatively referred to as apolypeptide. When the amino acids are α-amino acids, either theL-optical isomer or the D-optical isomer can be used. Additionally,unnatural amino acids, for example, β-alanine, phenylglycine andhomoarginine are also included. Commonly encountered amino acids thatare not gene-encoded may also be used in the present invention. All ofthe amino acids used in the present invention may be either the D- orL-isomer. The L-isomers are generally preferred. In addition, otherpeptidomimetics are also useful in the present invention. For a generalreview, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINOACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker, NewYork, p. 267 (1983).

“Bioactive species,” refers to molecules that, when administered to anorganism, affect that organism. Exemplary bioactive species includepharmaceuticals, pesticides, herbicides, growth regulators and the like.Bioactive species encompasses small molecules (i.e., approximately <1000daltons), oligomers, polymers and the like. Also included are nucleicacids and their analogues, peptides and their analogues and the like.

“Carrier molecule,” as used herein refers to any molecule to which acompound of the invention is attached. Representative carrier moleculesinclude a protein (e.g., enzyme, antibody), glycoprotein, peptide,saccharide (e.g., mono-oliogo- and poly-saccharides), hormone, receptor,antigen, substrate, metabolite, transition state analog, cofactor,inhibitor, drug, dye, nutrient, growth factor, etc., without limitation.“Carrier molecule” also refers to species that might not be consideredto fall within the classical definition of “a molecule,” e.g., solidsupport (e.g., synthesis support, chromatographic support, membrane),virus and microorganism.

“Activated derivatives of hydroxyl moieties,” and equivalent species,refer to compounds in which an oxygen-containing leaving group isformally derived from a hydroxyl moiety.

“Activated derivatives of carboxyl moieties,” and equivalent species,refer to compounds in which an oxygen-containing leaving group isformally derived from a carboxyl moiety.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals, having the number of carbon atoms designated (i.e.C₁-C₁₀ means one to ten carbons). Examples of saturated alkyl radicalsinclude, but are not limited to, groups such as methyl, methylene,ethyl, ethylene, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, includes “alkylene” and thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups, which are limited to hydrocarbon groups,are termed “homoalkyl”.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Also included aredi- and multi-valent species such as “cycloalkylene.” Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to, speciessuch as trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings), which are fused togetheror linked covalently. The term “heteroaryl” refers to aryl groups (orrings) that contain from one to four heteroatoms selected from N, O, andS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. A heteroaryl group canbe attached to the remainder of the molecule through a heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Also included are di- and multi-valentlinker species, such as “arylene.” Substituents for each of the abovenoted aryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) (“alkyl group substituents”) can be one or more of avariety of groups selected from, but not limited to: —OR′, ═O, ═NR′,═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′,—CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂ in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R′, R″, R′″ andR″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., arylsubstituted with 1-3 halogens, substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R″is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups (“aryl groupsubstituents”) are varied and are selected from, for example: halogen,—OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′,—C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR″C(O)₂R′, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R″″are preferably independently selected from hydrogen, (C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl and heteroaryl, (unsubstitutedaryl)-(C₁-C₄)alkyl, and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

“Analyte,” “target,” “substance to be assayed”, and “target species,” asutilized herein refer to the species of interest in an assay mixture.The terms refer to a substance, which is detected qualitatively orquantitatively using a material, process or device of the presentinvention. Examples of such substances include cells and portionsthereof, enzymes, antibodies, antibody fragments and other biomolecules,e.g., antigens, polypeptides, glycoproteins, polysaccharides, complexglycolipids, nucleic acids, effector molecules, receptor molecules,enzymes, inhibitors and the like and drugs, pesticides, herbicides,agents of war and other bioactive agents.

More illustratively, such substances include, but are not limited to,tumor markers such as α-fetoprotein, carcinoembryonic antigen (CEA), CA125, CA 19-9 and the like; various proteins, glycoproteins and complexglycolipids such as β₂-microglobulin (β₂ m), ferritin and the like;various hormones such as estradiol (E₂), estriol (E₃), human chorionicgonadotropin (hCG), luteinizing hormone (LH), human placental lactogen(hPL) and the like; various virus-related antigens and virus-relatedantibody molecules such as HBs antigen, anti-HBs antibody, HBc antigen,anti-HBc antibody, anti-HCV antibody, anti-HIV antibody and the like;various allergens and their corresponding IgE antibody molecules;narcotic drugs and medical drugs and metabolic products thereof; andnucleic acids having virus- and tumor-related polynucleotide sequences.

The term, “assay mixture,” refers to a mixture that includes the analyteand other components. The other components are, for example, diluents,buffers, detergents, and contaminating species, debris and the like thatare found mixed with the target. Illustrative examples include urine,sera, blood plasma, total blood, saliva, tear fluid, cerebrospinalfluid, secretory fluids from nipples and the like. Also included aresolid, gel or sol substances such as mucus, body tissues, cells and thelike suspended or dissolved in liquid materials such as buffers,extractants, solvents and the like.

The term “drug” or “pharmaceutical agent,” refers to bioactive compoundsthat cause an effect in a biological organism. Drugs used as affinitymoieties or targets can be neutral or in their salt forms. Moreover, thecompounds can be used in the present method in a prodrug form. Prodrugsare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of interest in thepresent invention.

Introduction

The present invention provides a class of reactive fluorescent compoundsthat are based upon the xanthene nucleus. Also provided are conjugatesof the xanthene dyes with carrier molecules, including biological,non-biological and biologically active species. Selected xanthene labelsdescribed herein include a functionalized linker arm that is readilyconverted into an array of reactive derivatives without requiring amodification of the xanthene nucleus. Accordingly, the compounds of theinvention provide an as yet undisclosed advantage, allowing facileaccess to an array of conjugates between the linker arm-derivatizedxanthene nucleus and carrier molecules.

Residing in the field of fluorescent labels, the present inventionprovides benefits of particular note. Fluorescent labels have theadvantage of requiring few precautions in handling, and being amenableto high-throughput visualization techniques (optical analysis includingdigitization of the image for analysis in an integrated systemcomprising a computer). Exemplary labels exhibit one or more of thefollowing characteristics: high sensitivity, high stability, lowbackground, low environmental sensitivity and high specificity inlabeling. Many fluorescent labels based upon the xanthene nucleus arecommercially available from the SIGMA chemical company (Saint Louis,Mo.), Molecular Probes (Eugene, Oreg.), R&D systems (Minneapolis,Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., AldrichChemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL LifeTechnologies, Inc. (Gaithersburg, Md.), Fluka Chemica-BiochemikaAnalytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems(Foster City, Calif.), as well as many other commercial sources known toone of skill. Furthermore, those of skill in the art will recognize howto select an appropriate xanthene-based fluorophore for a particularapplication and, if it not readily available commercially, will be ableto synthesize the necessary fluorophore de novo or synthetically modifycommercially available xanthene compounds to arrive at the desiredfluorescent label.

The compounds, probes and methods discussed in the following sectionsare generally representative of the compositions of the invention andthe methods in which such compositions can be used. The followingdiscussion is intended as illustrative of selected aspects andembodiments of the present invention and it should not be interpreted aslimiting the scope of the present invention.

DESCRIPTION OF THE EMBODIMENTS

In a first aspect, the present invention provides a fluorescent compoundhaving the formula:

in which R¹, R² and R⁴-R¹¹ are each independently selected from groupssuch as substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,halogen, H, NO₂, CN and C(Z¹)R¹⁴, NR¹⁵R¹⁶ and Z²R¹⁶. R³ is selected fromZ²R¹⁶ and NR¹⁵R¹⁶.

Z¹ represents O, S or NH. Z² is either O or S. Groups corresponding toR¹⁵ include H, substituted or unsubstituted alkyl, and substituted orunsubstituted heteroalkyl. R¹⁶ is selected from H, substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl andC(Z³)R¹⁷. R¹⁵ and R¹⁶, together with the nitrogen to which they areattached, can also be any nitrogen-containing reactive group. Exemplarygroups include —NHNH₂, —N═C═S and —N═C═O.

Z³ represents O, S or NH. The symbol R¹⁷ represents groups such assubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, OR¹⁸, and NR¹⁹R²⁰. R¹⁸ represents H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and C(O)R²¹. R¹⁹ and R²⁰ are symbols representing groupsindependently selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl. R²¹ is substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl.

The symbol Y represents either C(O) or S(O)₂. X is (NR²²R²³) or (O). R²²and R²³ are independently selected and are members selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl.

R¹² or R¹³ are independently selected from substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl, with the proviso that at leastone of R¹² or R¹³ includes an oxygen-containing reactive group or acarrier molecule to which the dye of the invention is conjugated througha moiety produced by reaction of the oxygen-containing reactive groupwith a reactive group on the carrier molecule of complementaryreactivity. R¹² and R¹³, together with the nitrogen atom to which theyare attached are optionally joined to form a ring. Exemplary ringsinclude 4-8-membered heterocylic and heteroaryl rings. When R¹² and R¹³,together with the nitrogen to which they are attached (“first nitrogen”)form a piperazine ring in which the second nitrogen atom isfunctionalized with an alkyl moiety bearing an oxygen-containingreactive group, the reactive group is preferably a phosphoramidite. Apiperazinyl-xanthene can be conjugated to another species, e.g., anucleic acid, saccharide, or a lipid, through oxygen-containing reactivegroups other than a phosphoramidite, e.g, an activated carboxylic acid,a carbonate, a reactive hydroxyl moiety, i.e., —OH, and R—X in which Ris a linker or bond to the xanthene and —X is a leaving group, e.g., asulfonate, or halide.

The substituents on the aryl ring nuclei can be joined to form rings.For example, in one embodiment in which R³ is NR¹⁵R¹⁶, R², R⁴ and R¹⁵and R¹⁶, together with the nitrogen atom to which they are bound, arefused with the phenyl moiety to which NR¹⁵R¹⁶, R² and R⁴ are bound,forming a substituted or unsubstituted ring system having the generalformula:

In yet another embodiment in which X is NR²²R²³, R⁵, R⁶ and R²² and R²³,together with the nitrogen atom to which they are bound, are fused withthe unsaturated 6-member ring to which NR²²R²³, R⁵ and R⁶ are bound,forming a substituted or unsubstituted ring system having the generalformula:

The present invention also provides a conjugate between a carriermolecule, e.g., a nucleic acid, and a fluorescent compound of theinvention, which is covalently or ionically bound to a moiety of thecarrier molecule. When the carrier molecule is a nucleic acid,representative moieties at which the fluorescent compounds of theinvention are attached include the sugar moiety, at O- and/or C-centers;endo- and/or exo-cyclic amines, carbon atoms of nucleobase moieties, andinternucleotide bridges. In still a further exemplary embodiment, theconjugate between the compound of the invention and the carrier moleculeincludes at least one moiety that quenches the fluorescence emission ofthe compound of the invention.

The compounds of the invention include a linker moiety as a component ofeither or both R¹² and R¹³. An exemplary linker is a substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl moietythat includes a reactive group at the terminus of the linker. In theconjugates of the invention, the reactive group is converted to alinking moiety by reaction with a group of complementary reactivity onthe species to which the dye is conjugated.

In a representative embodiment, the invention provides a xanthene dye,as set forth above, in which R¹² and/or R¹³ includes a linker arm moietyhaving the formula:

wherein L¹, L² and L³ are members independently selected from a bond,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. The index “t” is 0 or 1. Exemplary linker arms according tothis formula include one or more amide, urethane, ether, ester, urea,sulfonamide, sulfoxide, amine, sulfide, phosphate, or keto moiety. Inanother exemplary linker of use in the invention, one or more of L¹, L²or L³ includes from 1 to 6 ethylene glycol moieties. The points ofattachment shown above represent members independently selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl, a hydroxyl-protecting group, a phosphate moiety, aphosphodiester moiety, a phosphorus-containing internucleotide bridge, asolid support, a carrier molecule and a reactive functional group, e.g.,—OP(O)(OR^(o))(N(R^(p)R^(q)))₂. The groups represented by the symbolsR^(o), R^(p) and R^(q) are members independently selected from H,substituted or unsubstituted C₁-C₆ alkyl and substituted orunsubstituted C₁-C₆ heteroalkyl; and the index “s” is an integer from 1to 20. In an exemplary embodiment, R^(o) is CH₂CH₂CN.

A subset of R¹² and R¹³ moieties according to the motif set forth abovehas the formula:

in which the symbols R^(x) and R^(y) represent groups that areindependently selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl, a hydroxyl-protecting group, aphosphate moiety, a phosphodiester moiety, a phosphorus-containinginternucleotide bridge, a solid support, a carrier molecule and areactive functional group, e.g., —OP(O)(OR^(o))(N(R^(p)R^(q)))₂. Thegroups represented by the symbols R^(o), R^(p) and R^(q) are membersindependently selected from H, substituted or unsubstituted C₁-C₆ alkyland substituted or unsubstituted C₁-C₆ heteroalkyl; and the index “s” isan integer from 1 to 20. In an exemplary embodiment, R^(o) is CH₂CH₂CN.

The invention also provides fluorescent compounds in which at least oneof R^(x) and R^(y) comprises a moiety having the formula:

L⁴ is a member selected from a bond, substituted or unsubstituted alkyland substituted or unsubstituted heteroalkyl; and R^(z) is a memberselected from a reactive functional group, solid support, or a carriermolecule, e.g., a nucleic acid, a saccharide and a peptide.

In selected compounds of the invention, L⁴ comprises a moiety having theformula:

wherein the symbol Z³ represents either CH₂ or C═O.

In another embodiment, the invention provides xanthene dyes in which oneof the substituents on the xanthene nucleus, preferably R¹², includes amoiety having the structure:

in which L^(1a) is a member selected from substituted or unsubstitutedalkyl, and substituted or unsubstituted heteroalkyl groups. The symbolsR^(2a) and R^(3a) represent groups that are independently selected fromH, substituted or unsubstituted alkyl, and substituted or unsubstitutedheteroalkyl. The groups R² and R³, together with the nitrogen to whichthey are attached, are optionally joined to form a ring. Preferred ringstructures include substituted or unsubstituted C₅-C₇ cycloalkyl andsubstituted or unsubstituted 5-7-membered heterocycloalkyl.

An exemplary linker species according to the motif presented aboveincludes an NR¹²R¹³ moiety that has the formula:

in which R^(2a) and R^(3a) are members independently selected fromsubstituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. The symbols X^(2a) and X^(3a) represent groups that areindependently selected from H, substituted or unsubstituted lower alkyl,substituted or unsubstituted heteroalkyl, reactive functional groups, abond to a solid support and a bond to a carrier molecule. When thecarrier molecule is a nucleic acid, the bond can be to a nucleobase(e.g., to C or N), sugar (e.g., to C or O) or internucleotide bridge(e.g., to P, O, S, C or N).

Exemplary identities for X^(2a) and X^(3a) include —CH₃, —OX, —COOX,—NHX, —SX, halogen and —OP(O)(OR^(o))(N(R^(p)R^(q)))₂. X is H or anactivating group, e.g., N-hydroxysuccinimide, or sulfonate ester. Thegroups represented by the symbols R^(o), R^(p) and R^(q) are membersindependently selected from H, substituted or unsubstituted C₁-C₆ alkyland substituted or unsubstituted C₁-C₆ heteroalkyl; and the index “s” isan integer from 1 to 20. In an exemplary embodiment, R^(o) is CH₂CH₂CN.

When X^(2a) or X^(3a) is a component of a linkage between a species ofthe invention and a carrier molecule or solid support, it is modified ina manner that satisfies the rules of valence, e.g, —OH becomes —O—; COOHbecomes COOR, CONRR′, etc.

In another preferred embodiment, a member selected from R^(2a), R^(3a)and combinations thereof comprises a polyether. Preferred polyethersinclude, for example, poly(ethylene glycol), poly(propyleneglycol) andcopolymers thereof. The polyether may be internal to the R^(2a) orR^(3a) group or it may form the free terminus of the group. When thepolyether is at the terminus of the group, the terminal —O— moiety ispresent as —OH, alkoxy or one of a variety of the groups referred toherein as substituents for alkyl moieties. See, for example, ShearwaterPolymers, Inc. Catalog of Poly(ethylene glycol) Derivatives 2002.

In a further exemplary embodiment, NR¹²R¹³ has the formula:

in which the indexes p and q are integers independently selected from 1to 20, inclusive, preferably from 2 to 16, inclusive. X^(2a) and X^(3a)are as described above.

In yet another exemplary embodiment, NR¹²R¹³ has the formula:

in which the index “v” is 0 or 1. R^(2a) and R^(3a) are independentlyselected from a bond, substituted or unsubstituted alkyl or substitutedor unsubstituted heteroalkyl moieties (e.g., ethers, polyethers). When“v” is 0, the resulting phosphate in the structure above canalternatively be OH (i.e., 3′- or 5′-hydroxyl). In other words, althoughrepresented as interposed between two nucleotides, the fluorescent labelof the invention can be placed at any point between two nucleoside ornucleotide subunits in a nucleic acid. Thus, exemplary compounds includeNR¹²R¹³ at an internal position of the nucleic acid. In other exemplaryembodiments, NR¹²R¹³ is tethered to the nucleic acid at the linkagebetween the 5′ and 5′-1 residues and/or the linkage between the 3′ and3′-1 residues.

In a further exemplary embodiment, NR¹²R¹³ has the formula:

When “v” is 0, the resulting phosphate in the structure above canalternatively be OH (i.e., 3″- or 5′-hydroxyl).

The invention also provides nucleic acid derivatives in which a compoundof the invention is conjugated to a sugar moiety of the nucleic acid. Anexemplary species according to this motif has the formula:

in which the index u and the index g independently represent 0, 1 or anumber greater than one, with the proviso that at least one of u and gis preferably non-zero. Although shown attached to the 2′ carbon of the3′-terminus of the nucleic acid, those of skill will appreciate that asimilar structure tethered to the 5′-terminus, or an internal site ofthe nucleic acid is within the scope of the invention. Moreover, thegroup can be tethered through the O atom of a 2′-hydroxyl. When “u” is0, the phosphate/phosphodiester group is optionally OH.

Moreover, the agents of the invention can be conjugated through the 3′-and/or 5′-hydroxyl moiety of a nucleic acid.

In another exemplary embodiment, NR¹²R¹³ has the formula:

in which h and i are members independently selected from integers suchthat the sum (h+i) is from 4-8. R²⁵ is a reactive functional group,e.g., an oxygen-containing reactive functional group, or a substitutedor unsubstituted alkyl or substituted or unsubstituted heteroalkylmoiety bearing an oxygen-containing reactive functional group. In anexemplary embodiment, R²⁵ is a phosphoramidite or a species thatincludes a phosphoramidite. In a further exemplary embodiment, h and iare both 2.

Representative compounds of the invention are set forth in FIG. 1.

Synthesis

The compounds of the invention are synthesized by an appropriatecombination of generally well-known synthetic methods. Techniques usefulin synthesizing the compounds of the invention are both readily apparentand accessible to those of skill in the relevant art. The discussionbelow is offered to illustrate certain of the diverse methods availablefor use in assembling the compounds of the invention, it is not intendedto define the scope of reactions or reaction sequences that are usefulin preparing the compounds of the present invention.

The compounds of the invention can be prepared as a single isomer or amixture of isomers, including, for example cis-isomers, trans-isomers,diastereomers and stereoisomers. In a preferred embodiment, thecompounds are prepared as substantially a single isomer. Isomericallypure compounds are prepared by using synthetic intermediates that areisomerically pure in combination with reactions that either leave thestereochemistry at a chiral center unchanged or result in its completeinversion. Alternatively, the final product or intermediates along thesynthetic route can be resolved into a single isomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose anappropriate resolution or synthetic method for a particular situation.See, generally, Furniss et al. (eds.) VOGEL'S ENCYCLOPEDIA OF PRACTICALORGANIC CHEMISTRY 5^(TH) ED., Longman Scientific and Technical Ltd.,Essex, 1991, pp. 809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

An exemplary synthetic route to compounds of the invention is set forthin Scheme 1. Rhodamine B acid chloride 1 is prepared by the action ofphosphorus oxychloride on the precursor dye carboxylic acid. Theactivated dye is reacted with selected aminoalcohols to formcorresponding amides 2 and 5. The linker arm is readily extended byactivating the hydroxyl moiety of 5 and forming urethane 7 with anappropriate amino alcohol. The dyes bearing the hydroxyl terminatedlinker arms are converted to the corresponding phosphoramidites 3, 6 and8.

Scheme 2 outlines the preparation of a series of linker arm derivatizedxanthene dyes of the invention. The ethyl ester of Rhodamine 6G washydrolyzed in base, affording the corresponding acid 9, which wasactivated as the N-hydroxysuccinimide ester 10 and converted to thecorresponding hydroxyl-terminated alkyl amide 11. Alternatively, theester is aminolysed with an aminoalcohol, producing amide 12. The amideis activated as the p-nitrophenylchloroformate 13 and coupled with6-amino-1-hexanol, affording 14, which is readily converted to thecorresponding phosphoramidite 15.

Scheme 3 sets out an exemplary route for preparing a linker armderivatized dye and the corresponding phosphoramidite from rhodamine101. The dye-linker arm conjugate 17 is formed by aminolysis of ester 16with N-methylaminoethanol. The p-nitrophenylcarbonate activated analogueof 17 is reacted with 6-amino-1-hexanol, providing urethane 18, which isconverted to phosphoramidite 19.

In Scheme 4, ethyl ester 20 is formed by the action ofchlorotrimethylsilane and ethanol on the parent compound. Aminolysis ofthe ester with N-methylaminoethanol affords amide 21. Protection of thehydroxyl moiety as the DMT ether provides 22, and protection of thephenol-like hydroxyl as the t-butylacetyl group results in 23. The DMTmoiety is removed with dichloroacetic acid and the resulting alcohol 24is converted to the phosphoramidite 25.

As shown in Scheme 5, p-nitrophenylcarbonate 13 is converted to urethane26 withN⁴-(2-(4,7,10-trioxa-1,13-tridecanediamine)-5-methyl-5′-(4,4′-dimethoxytrityl)-3′-O-tert-butyldimethylsilyl-2′deoxycytidine.The TBDMS group is removed from the urethane and the resulting product27 is reacted with diglycolic anhydride to afford the acid 28 which isconjugated to activated controlled pore glass, affording 29.

Each of the phosphoramidites described above and the CPG-immobilizedconjugate 29 are of use in the synthesis of nucleic acids. Thephosphoramidites are cleanly coupled to a nucleic acid as demonstratedby chromatographic analysis of TTCGATAAGTCTAG, labeled at the 5′-Gmoiety with a compound of the invention.

For example, FIG. 2 is a reverse phase HPLC trace of a conjugate betweenthe nucleic acid and 15, showing the purity of the conjugate. The massspectrum, which shows a peak corresponding to the conjugate (M/e 5214),is shown in FIG. 3. FIG. 4 is an anion exchange HPLC chromatogram of aconjugate between the nucleic acid and 25. The mass spectrum (FIG. 5) ofthe nucleic acid conjugate with 25 includes a major peak at M/e 5128,corresponding to the conjugate. The reverse-phase HPLC chromatogram ofthe conjugate between the nucleic acid and 19 is shown in FIG. 6. A massspectrum of this conjugate has a peak (M/e 5304) corresponding to themolecular weight of the conjugate (FIG. 7). The anion exchangechromatogram of the conjugate between the nucleic acid and 3′-OH linked28 is shown in FIG. 8 and the mass spectrum in FIG. 9.

The absorption and emission profiles of conjugates of the xanthenes ofthe invention 25, 15, 6 and 19 and a model nucleic acid (TTTTTTTTTTT)are shown in FIG. 10, FIG. 11, FIG. 12, FIG. 13, respectively.

Chemical synthesis of the nucleic acid is generally automated and it isperformed by coupling nucleosides through phosphorus-containing covalentlinkages. The most commonly used oligonucleotide synthesis methodinvolves reacting a nucleoside with a protected cyanoethylphosphoramidite monomer in the presence of a weak acid. The couplingstep is followed by oxidation of the resulting phosphite linkage.Finally, the cyanoethyl protecting group is removed and the nucleic acidis cleaved from the solid support on which it was synthesized. Thelabels of the present invention can be incorporated duringoligonucleotide synthesis using a phosphoramidite derivative of thefluorescent compound of the invention. Alternatively, the label can beintroduced by combining a compound of the invention that includes areactive functional group with the nucleic acid under appropriateconditions to couple the compound to the nucleic acid. In yet anotherembodiment, the fluorescent compound is attached to a solid supportthrough a linker arm, such as a substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl or a nucleic acid residue.Synthesis proceeds with the fluorescent moiety already in place on thegrowing nucleic acid chain.

Enzymatic methods of synthesis involve the use of fluorescent-labelednucleic acids in conjunction with a nucleic acid template, a primer andan enzyme. Efficient enzymatic incorporation of a fluorescent-labelednucleic acid is facilitated by selection of reaction partners that donot adversely affect the enzymes ability to couple the partners.

In those embodiments of the invention in which the xanthene-basedfluorescent compound of the invention is attached to a nucleic acid, thecarrier molecule is produced by either synthetic (solid phase, liquidphase or a combination) or enzymatically, or by a combination of theseprocesses. One synthetic strategy for the preparation ofoligonucleotides is the H-phosphonate method (Froehler, B. andMatteucci, M. (1986) Tetrahedron Lett., (27): 469-472). This methodutilizes activated nucleoside H-phosphonate monomers rather thanphosphoramidites to create the phosphate internucleotide linkage. Incontrast to the phosphoramidite method, the resulting phosphonatelinkage does not require oxidation every cycle but instead only a singleoxidation step at the end of chain assembly. The H-phosphonate methodmay also be used to conjugate reporters and dyes to syntheticoligonucleotide chains (Sinha, N. and Cook, R. (1988) Nucleic AcidsResearch, (16): 2659).

The four bases of the nucleosides, adenine, thymine (uracil in RNA),guanine and cytosine include moieties that are chemically reactive(e.g., exocyclic amines) and must be protected from participating inundesirable reactions during the synthesis of the oligonucleotide by“blocking” the reactive sites with a moiety that can be removed once thesynthesis is complete. Traditional protecting groups include the benzoyl(dA, dC) and isobutyryl (dC, dG) groups. Each of the aforementionedprotecting groups is base-labile and is typically cleaved, withconcomitant cleavage of the oligonucleotide from the synthesis support,using ammonia/water mixtures. A full review of protecting groups may befound in “Advances in the Synthesis of Oligonucleotides by thePhosphoramidite Approach”, Tetrahedron Vol 48, p 2223-2311, 1992).

In another exemplary embodiment, the invention provides a method ofdeprotecting a conjugate formed between a xanthene dye of the inventionand a nucleic acid. The method consists of contacting a precursor to theconjugate in which the nucleic acid monomers are protected with adeprotection cocktail that includes an amine and alcohol. It has beendiscovered that use the deprotection cocktail with the conjugates of theinvention significantly enhances the yield of the deprotected conjugate,minimizing degradation of the xanthene dye during deprotection.

The oligonucleotide may be cleaved from the solid support beforedeprotection, after deprotection or concomitant with deprotection. Thus,in another embodiment, the oligonucleotide is tethered to a solidsupport via a linker arm that is not cleaved by the organic amine thatremoves the base-labile protecting group. In this embodiment, theoligonucleotide may be cleaved from the support using a reagent otherthan the deprotection reagent. Thus, the method of the invention furtherincludes the step of contacting the support-bound oligonucleotide with acleavage reagent, thereby forming a cleavage mixture, and incubating thecleavage mixture for a period of time sufficient to cleave theoligonucleotide from the support.

The superior flexibility of the method of the invention allows virtuallyany primary or secondary organic amine to be of use as the deprotectingagent. The amines set forth below are merely exemplary, and those ofskill will appreciate that the invention is not limited to the use ofthe explicitly recited amines. Amines of use in practicing the presentinvention include substituted or unsubstituted alkyl amines, substitutedor unsubstituted aryl amines, substituted or unsubstituted heteroarylamines and substituted or unsubstituted heterocyclic amines.

Also useful in the method of the invention are the alkyl amino alcohols,e.g., aminoethanol, aminopropanol, 2-amino-2-methyl-1-propanol andcombinations thereof. Alkyl amino ethers, e.g.,1,4-butanediol-bis-(3-aminopropyl)ether,4,7,10-trioxa-1,13-tridecanamine and combinations thereof can also beused. The amine may also be a polymeric amine, e.g, poly(ethyleneimine),poly(ethylene glycol)amine and combinations thereof. Other amines andcombinations of amines will be apparent to those of skill in the art.

Reactive Functional Groups

The compounds of the invention bear a reactive functional group, whichcan be located at any position on the molecule. Exemplary speciesinclude a reactive functional group as a constituent of at least one ofR¹² and R¹³. Exemplary reactive groups are members independentlyselected from substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl, with the proviso that at least one of R¹² orR¹³ includes an oxygen-containing reactive group or a carrier molecule.When the reactive group is attached a substituted or unsubstituted alkylor substituted or unsubstituted heteroalkyl moiety, the reactive groupis preferably located at a terminal position of the alkyl or heteroalkylchain. Reactive groups and classes of reactions useful in practicing thepresent invention are generally those that are well known in the art ofbioconjugate chemistry. Currently favored classes of reactions availablewith reactive xanthene-based compounds of the invention are thoseproceeding under relatively mild conditions. These include, but are notlimited to nucleophilic substitutions (e.g., reactions of amines andalcohols with acyl halides, active esters), electrophilic substitutions(e.g., enamine reactions) and additions to carbon-carbon andcarbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alderaddition). These and other useful reactions are discussed in, forexample, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons,New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, SanDiego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances inChemistry Series, Vol. 198, American Chemical Society, Washington, D.C.,1982.

Useful reactive functional groups include, for example:

(a) carboxyl groups and derivatives thereof including, but not limitedto activated esters, e.g., N-hydroxysuccinimide esters,N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters,p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters,activating groups used in peptide synthesis and acid halides;

(b) hydroxyl groups, which can be converted to esters, sulfonates,phosphoramidates, ethers, aldehydes, etc.

(c) haloalkyl groups, wherein the halide can be displaced with anucleophilic group such as, for example, an amine, a carboxylate anion,thiol anion, carbanion, or an alkoxide ion, thereby resulting in thecovalent attachment of a new group at the site of the halogen atom;

(d) dienophile groups, which are capable of participating in Diels-Alderreactions such as, for example, maleimido groups;

(e) aldehyde or ketone groups, allowing derivatization via formation ofcarbonyl derivatives, e.g., imines, hydrazones, semicarbazones oroximes, or via such mechanisms as Grignard addition or alkyllithiumaddition;

(f) sulfonyl halide groups for reaction with amines, for example, toform sulfonamides;

(g) thiol groups, which can be converted to disulfides or reacted withacyl halides, for example;

(h) amine or sulfhydryl groups, which can be, for example, acylated,alkylated or oxidized;

(i) alkenes, which can undergo, for example, cycloadditions, acylation,Michael addition, etc;

(j) epoxides, which can react with, for example, amines and hydroxylcompounds; and

(k) phosphoramidites and other standard functional groups useful innucleic acid synthesis.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assembleor utilize the reactive xanthene analogue. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

In addition to those embodiments in which a compound of the invention isattached directly to a carrier molecule, the fluorophores can also beattached by indirect means. In this embodiment, a ligand molecule (e.g.,biotin) is generally covalently bound to the probe species. The ligandthen binds to another molecules (e.g., streptavidin) molecule, which iseither inherently detectable or covalently bound to a signal system,such as a fluorescent compound, or an enzyme that produces a fluorescentcompound by conversion of a non-fluorescent compound. Useful enzymes ofinterest as labels include, for example, hydrolases, particularlyphosphatases, esterases and glycosidases, hydrolases, peptidases oroxidases, and peroxidases.

Probes

The invention provides probes having a xanthene dye of the inventionconjugated to a carrier molecule, for example, a target species (e.g.,receptor, enzyme, etc.) a ligand for a target species (e.g., nucleicacid, peptide, etc.), a small molecule (e.g., drug, pesticide, etc.), asolid support and the like. The probes can be used for in vitro and invivo applications.

An unexpected property of the xanthene dyes of the invention is theirrobustness under a variety of synthetic conditions used to attach thexanthene dye of the invention to a carrier molecule. For example, manyof the xanthene dyes of the invention survive the conditions necessaryfor automated synthesis of nucleic acids without undergoing anysubstantial degree of degradation or alteration. In contrast, many ofart-recognized fluorophores presently in use require the use of specialconditions to assemble the carrier molecule to which they are attached,or they have to be attached after the completion of the carrier moleculesynthesis. The additional complexity of the synthesis of a probeincreases both the duration of the synthesis and its cost.

Dual Labeled Probes

The present invention also provides dual labeled probes that includeboth a xanthene dye of the invention and second label. Exemplary secondlabels include quenchers of fluorescence energy, such as that emitted bythe xanthene dyes of the invention. Quenchers of use in the dual labeledprobes of the invention are known to those of skill in the art. See, forexample, commonly owned PCT application U.S. 01/15082 disclosing, “BlackHole” quenchers. A preferred quencher for use with the xanthenes of theinvention comprises a diazo bond between two radicals independentlyselected from substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl groups. The quencher is generally conjugated tothe xanthene or to a linker attached to the xanthene.

The dual labeled probes include a first and a second probe attached to astructure linking the two labels. Of note are linkers that arebiomolecules, e.g., nucleic acids, peptides, lipids, saccharides and thelike; drug moieties; substituted and unsubstituted alkyl, substitutedand unsubstituted heteroalkyl, substituted and unsubstituted aryl,substituted and unsubstituted heteroaryl and substituted andunsubstituted heterocycloalkyl moieties. Selected linkers of use withthe xanthenes of the invention are discussed in greater detail herein.

Exemplary dual labeled probes include nucleic acid probes that include anucleic acid with a xanthene dye of the invention attached thereto.Exemplary probes include both a xanthene dye of the invention and aquencher. The probes are of use in a variety of assay formats. Forexample, when a nucleic acid singly labeled with a xanthene dye of theinvention is the probe, the interaction between the first and secondnucleic acids can be detected by observing the interaction between thexanthene dye of the invention and the nucleic acid. Alternatively, theinteraction is the quenching by a quencher attached to the secondnucleic acid of the fluorescence from a xanthene dye of the invention.

The xanthene dyes of the invention are useful in conjunction withnucleic-acid probes in a variety of nucleic acidamplification/quantification strategies including, for example,5′-nuclease assay, Strand Displacement Amplification (SDA), Nucleic AcidSequence-Based Amplification (NASBA), Rolling Circle Amplification(RCA), as well as for direct detection of targets in solution phase orsolid phase (e.g., array) assays. Furthermore, the xanthene dye of theinvention-derivatized nucleic acids can be used in probes ofsubstantially any format, including, for example, format selected frommolecular beacons, Scorpion Probes™, Sunrise Probes™, conformationallyassisted probes, light up probes, Invader Detection probes, and TaqMan™probes. See, for example, Cardullo, R., et al., Proc. Natl. Acad. Sci.USA, 85:8790-8794 (1988); Dexter, D. L., J. Chem. Physics, 21:836-850(1953); Hochstrasser, R. A., et al., Biophysical Chemistry, 45:133-141(1992); Selvin, P., Methods in Enzymology, 246:300-334 (1995);Steinberg, I., Ann. Rev. Biochem., 40:83-114 (1971); Stryer, L., Ann.Rev. Biochem., 47:819-846 (1978); Wang, G., et al., Tetrahedron Letters,31:6493-6496 (1990); Wang, Y., et al., Anal. Chem., 67:1197-1203 (1995);Debouck, C., et al., in supplement to nature genetics, 21:48-50 (1999);Rehman, F. N., et al., Nucleic Acids Research, 27:649-655 (1999);Cooper, J. P., et al., Biochemistry, 29:9261-9268 (1990); Gibson, E. M.,et al., Genome Methods, 6:995-1001 (1996); Hochstrasser, R. A., et al.,Biophysical Chemistry, 45:133-141 (1992); Holland, P. M., et al., ProcNatl. Acad. Sci USA, 88:7276-7289 (1991); Lee, L. G., et al., NucleicAcids Rsch., 21:3761-3766 (1993); Livak, K. J., et al., PCR Methods andApplications, Cold Spring Harbor Press (1995); Vamosi, G., et al.,Biophysical Journal, 71:972-994 (1996); Wittwer, C. T., et al.,Biotechniques, 22:176-181 (1997); Wittwer, C. T., et al., Biotechniques,22:130-38 (1997); Giesendorf, B. A. J., et al., Clinical Chemistry,44:482-486 (1998); Kostrikis, L. G., et al., Science, 279:1228-1229(1998); Matsuo, T., Biochemica et Biophysica Acta, 1379:178-184 (1998);Piatek, A. S., et al., Nature Biotechnology, 16:359-363 (1998);Schofield, P., et al., Appl. Environ. Microbiology, 63:1143-1147 (1997);Tyagi S., et al., Nature Biotechnology, 16:49-53 (1998); Tyagi, S., etal., Nature Biotechnology, 14:303-308 (1996); Nazarenko, I. A., et al.,Nucleic Acids Research, 25:2516-2521 (1997); Uehara, H., et al.,Biotechniques, 26:552-558 (1999); D. Whitcombe, et al., NatureBiotechnology, 17:804-807 (1999); Lyamichev, V., et al., NatureBiotechnology, 17:292 (1999); Daubendiek, et al., Nature Biotechnology,15:273-277 (1997); Lizardi, P. M., et al., Nature Genetics, 19:225-232(1998); Walker, G., et al., Nucleic Acids Res., 20:1691-1696 (1992);Walker, G. T., et al., Clinical Chemistry, 42:9-13 (1996); and Compton,J., Nature, 350:91-92 (1991).

In view of the well-developed body of literature concerning theconjugation of small molecules to nucleic acids, many other methods ofattaching donor/acceptor pairs to nucleic acids will be apparent tothose of skill in the art.

More specifically, there are many linking moieties and methodologies forattaching groups to the 5′- or 3′-termini of nucleic acids, asexemplified by the following references: Eckstein, editor, Nucleic acidsand Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckermanet al., Nucleic Acids Research, 15: 5305-5321 (1987) (3′-thiol group onnucleic acid); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′-sulfhydryl); Giusti et al., PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′-phosphoamino groupvia Aminolink™ II available from P.E. Biosystems, CA.) Stabinsky, U.S.Pat. No. 4,739,044 (3-aminoalkylphosphoryl group); Agrawal et al.,Tetrahedron Letters, 31: 1543-1546 (1990) (attachment viaphosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15:4837 (1987) (5-mercapto group); Nelson et al., Nucleic Acids Research,17: 7187-7194 (1989) (3′-amino group), and the like. Methods forattaching the dyes to other nucleic acid moieties, e.g., internucleotidebridges, sugar C-atoms, nucleobase atoms, etc. are known to those ofskill in the art.

Exemplary fluorophores that can be combined in a probe with a xanthenedye of the invention include those set forth in Table 1.

TABLE 1 Suitable moieties that can be selected as donors or acceptors indonor-acceptor energy transfer pairs4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid acridine andderivatives: acridine acridine isothiocyanate5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonateN-(4-anilino-1-naphthyl)maleimide anthranilamide BODIPY Brilliant Yellowcoumarin and derivatives: coumarin 7-amino-4-methylcoumarin (AMC,Coumarin 120) 7-amino-4-trifluoromethylcouluarin (Coumaran 151) xanthenedyes cyanosine 4′,6-diaminidino-2-phenylindole (DAPI)5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red)7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid5-[diN-methylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride)4-(4′-diN-methylaminophenylazo)benzoic acid (DABCYL)4-diN-methylaminophenylazophenyl-4′-isothiocyanate (DABITC) eosin andderivatives: eosin eosin isothiocyanate erythrosin and derivatives:erythrosin B erythrosin isothiocyanate ethidium fluorescein andderivatives: 5-carboxyfluorescein (FAM)5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE) fluoresceinfluorescein isothiocyanate QFITC (XRITC) fluorescamine IR144 IR1446Malachite Green isothiocyanate 4-methylumbelliferone orthocresolphthalein nitrotyrosine pararosaniline Phenol Red B-phycoerythrino-phthaldialdehyde pyrene and derivatives: pyrene pyrene butyratesuccinimidyl 1-pyrene butyrate quantum dots Reactive Red 4 (Cibacron ™Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine(ROX) 6-carboxyrhodamine (R6G) lissamine rhodamine B sulfonyl chloriderhodamine (Rhod) rhodamine B rhodamine 123 rhodamine X isothiocyanatesulforhodamine B sulforhodamine 101 sulfonyl chloride derivative ofsulforhodamine 101 (Texas Red) N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA) tetramethyl rhodamine tetramethyl rhodamine isothiocyanate(TRITC) riboflavin rosolic acid terbium chelate derivatives

There is a great deal of practical guidance available in the literaturefor selecting appropriate donor-acceptor pairs for particular probes, asexemplified by the following references: Pesce et al., Eds.,FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971); White et al.,FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York,1970); and the like. The literature also includes references providingexhaustive lists of fluorescent and chromogenic molecules and theirrelevant optical properties for choosing reporter-quencher pairs (see,for example, Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATICMOLECULES, 2nd Edition (Academic Press, New York, 1971); Griffiths,COLOUR AND CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York,1976); Bishop, Ed., INDICATORS (Pergamon Press, Oxford, 1972); Haugland,HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes,Eugene, 1992) Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (IntersciencePublishers, New York, 1949); and the like. Further, there is extensiveguidance in the literature for derivatizing reporter and quenchermolecules for covalent attachment via common reactive groups that can beadded to a nucleic acid, as exemplified by the following references:Haugland (supra); Ullman et al., U.S. Pat. No. 3,996,345; Khanna et al.,U.S. Pat. No. 4,351,760. Thus, it is well within the abilities of thoseof skill in the art to choose an energy exchange pair for a particularapplication and to conjugate the members of this pair to a probemolecule, such as, for example, a nucleic acid, peptide or otherpolymer.

As will be apparent to those of skill in the art the methods set forthabove are equally applicable to the coupling to a nucleic acid of groupsother than the fluorescent compounds of the invention, e.g., quenchers,intercalating agents, hybridization enhancing moieties, minor groovebinders, alkylating agents, cleaving agents, etc.

For example, in selected embodiments, the probe includes a metal chelateor a chelating agent attached to the carrier molecule. The use of thesecompounds to bind to specific compounds is well known to those of skillin the art. See, for example, Pitt et al. “The Design of ChelatingAgents for the Treatment of Iron Overload,” In, INORGANIC CHEMISTRY INBIOLOGY AND MEDICINE; Martell, A. E., Ed.; American Chemical Society,Washington, D.C., 1980, pp. 279-312; Lindoy, L. F., THE CHEMISTRY OFMACROCYCLIC LIGAND COMPLEXES; Cambridge University Press, Cambridge,1989; Dugas, H., BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989,and references contained therein.

Additionally, a manifold of routes allowing the attachment of chelatingagents, crown ethers and cyclodextrins to other molecules is availableto those of skill in the art. See, for example, Meares et al.,“Properties of In Vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, R. E., Whitaker, J. R., Eds., American ChemicalSociety, Washington, D.C., 1982, pp. 370-387; Kasina et al. BioconjugateChem. 9:108-117 (1998); Song et al., Bioconjugate Chem. 8:249-255(1997).

In a presently preferred embodiment, the chelating agent is apolyaminocarboxylate chelating agent such as ethylenediaminetetraaceticacid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). Thesechelating agents can be attached to any amine-terminated component of acarrier molecule or a spacer arm, for example, by utilizing thecommercially available dianhydride (Aldrich Chemical Co., Milwaukee,Wis.).

The nucleic acids for use in the probes of the invention can be anysuitable size, and are preferably in the range of from about 10 to about100 nucleotides, more preferably from about 10 to about 80 nucleotidesand more preferably still, from about 20 to about 40 nucleotides. Theprecise sequence and length of a nucleic acid probe of the inventiondepends in part on the nature of the target polynucleotide to which itbinds. The binding location and length may be varied to achieveappropriate annealing and melting properties for a particularembodiment. Guidance for making such design choices can be found in manyart-recognized references.

Preferably, the 3′-terminal nucleotide of the nucleic acid probe isblocked or rendered incapable of extension by a nucleic acid polymerase.Such blocking is conveniently carried out by the attachment of a donoror acceptor moiety to the terminal 3′-position of the nucleic acidprobe, either directly or by a linking moiety.

The nucleic acid can comprise DNA, RNA or chimeric mixtures orderivatives or modified versions thereof. Both the probe and targetnucleic acid can be present as a single strand, duplex, triplex, etc.Moreover, as discussed above, the nucleic acid can be modified at thebase moiety, sugar moiety, or phosphate backbone with other groups suchas radioactive labels, minor groove binders, intercalating agents, donorand/or acceptor moieties and the like.

For example, the nucleic acid can comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxyN-methylaminomethyl-2-thiouridine,5-carboxy-N-methylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-adenine,7-methylguanine, 5-N-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,nitroindole, and 2,6-diaminopurine. The xanthene dye of the invention oranother probe component can be attached to the modified base.

In another embodiment, the nucleic acid comprises at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose. The xanthene dye oranother probe component can be attached to the modified sugar moiety.

In yet another embodiment, the nucleic acid comprises at least onemodified phosphate backbone selected from the group including, but notlimited to, a peptide nucleic acid hybrid, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof. The xanthene dye or another probecomponent can be attached to the modified phosphate backbone.

Phosphodiester linked nucleic acids of the invention can be synthesizedby standard methods known in the art, e.g. by use of an automated DNAsynthesizer using commercially available amidite chemistries (Ozaki etal., Nucleic Acids Research, 20: 5205-5214 (1992); Agrawal et al.,Nucleic Acids Research, 18: 5419-5423 (1990); Beaucage et al.,Tetrahedron, 48: 2223-2311 (1992); Molko et al., U.S. Pat. No.4,980,460; Koster et al., U.S. Pat. No. 4,725,677; Caruthers et al.,U.S. Pat. Nos. 4,415,732; 4,458,066; and 4,973,679). Nucleic acidsbearing modified phosphodiester linking groups can be synthesized bymethods known in the art. For example, phosphorothioate nucleic acidsmay be synthesized by the method of Stein et al. (Nucl. Acids Res.16:3209 (1988)), methylphosphonate nucleic acids can be prepared by useof controlled pore glass polymer supports (Sarin et al., Proc. Natl.Acad. Sci. U.S.A. 85:7448-7451 (1988)). Other methods of synthesizingboth phosphodiester- and modified phosphodiester-linked nucleic acidswill be apparent to those of skill in the art.

When the nucleic acids are synthesized utilizing an automated nucleicacid synthesizer, the donor and acceptor moieties are preferablyintroduced during automated synthesis. Alternatively, one or more ofthese moieties can be introduced either before or after the automatedsynthesis procedure has commenced. For example, donor and/or acceptorgroups can be introduced at the 3′-terminus using a solid supportmodified with the desired group(s). Additionally, donor and/or acceptorgroups can be introduced at the 5′-terminus by, for example a derivativeof the group that includes a phosphoramidite. In another exemplaryembodiment, one or more of the donor and/or acceptor groups isintroduced after the automated synthesis is complete.

In the dual labeled probes, the quencher moiety is preferably separatedfrom the xanthene dye of the invention by at least about 6 nucleotides,more preferably at least about 10 nucleotides, and more preferably by atleast about 15 nucleotides. The quencher moiety is preferably attachedto either the 3′- or 5′-terminal nucleotides of the probe. The xanthenedye of the invention moiety is also preferably attached to either the3′- or 5′-terminal nucleotides of the probe. More preferably, the donorand acceptor moieties are attached to the 3′- and 5′- or 5′- and3′-terminal nucleotides of the probe, respectively, although internalplacement is also useful.

Once the desired nucleic acid is synthesized, it is preferably cleavedfrom the solid support on which it was synthesized and treated, bymethods known in the art, to remove any protecting groups present (e.g.,60° C., 5 h, concentrated ammonia). In those embodiments in which abase-sensitive group is attached to the nucleic acids (e.g., TAMRA), thedeprotection will preferably use milder conditions (e.g.,butylamine:water 1:3, 8 hours, 70° C.). Deprotection under theseconditions is facilitated by the use of quick deprotect amidites (e.g.,dC-acetyl, dG-dmf). An alternative work up utilizes 2-methoxyethylamineand methanol.

Following cleavage from the support and deprotection, the nucleic acidis purified by any method known in the art, including chromatography,extraction and gel purification. In a preferred embodiment, the nucleicacid is purified using HPLC. The concentration and purity of theisolated nucleic acid is preferably determined by measuring the opticaldensity at 260 nm in a spectrophotometer.

Exemplary dual labeled probes of the invention were prepared asdescribed in the Examples section.

Small Molecule Probes

The xanthene dyes of the invention can be used as components of smallmolecule probes. In a preferred design, a small molecule probe includesa xanthene dye of the invention and a second species that alters theluminescent properties of the dyes, e.g., a quencher of fluorescence. Inan exemplary embodiment, an agent such as an enzyme cleaves the xanthenedye of the invention, the quencher or both from the small moleculegenerating fluorescence in the system under investigation (see, forexample, Zlokarnik et al., Science 279: 84-88 (1998)).

Nucleic Acid Capture Probes

In one embodiment, an immobilized nucleic acid comprising a xanthene dyeof the invention is used as a capture probe. The nucleic acid probe canbe used in solution phase or it can be attached to a solid support. Theimmobilized probes can be attached directly to the solid support orthrough a linker arm between the support and the xanthene dye or betweenthe support and a nucleic acid residue. Preferably, the probe isattached to the solid support by a linker (i.e., spacer arm, supra). Thelinker serves to distance the probe from the solid support. The linkeris most preferably from about 5 to about 30 atoms in length, morepreferably from about 10 to about 50 atoms in length. Exemplaryattachment points include the 3′- or 5′-terminal nucleotide of the probeas well as other accessible sites discussed herein.

In yet another preferred embodiment, the solid support is also used asthe synthesis support in preparing the probe. The length and chemicalstability of the linker between the solid support and the first 3′-unitof nucleic acid (or the xanthene dye) play an important role inefficient synthesis and hybridization of support bound nucleic acids.The linker arm should be sufficiently long so that a high yield (>97%)can be achieved during automated synthesis. The required length of thelinker will depend on the particular solid support used. Exemplarylinkers are from about 6 to about 30 atoms in length. For nucleic acidsynthesis, the linker arm is usually attached to the 3′-OH of the3′-terminus by a cleaveable linkage, e.g., an ester linkage, which canbe cleaved with appropriate reagents to free the nucleic acid from thesolid support.

Hybridization of a probe immobilized on a solid support generallyrequires that the probe be separated from the solid support. A preferredlinker for this embodiment includes at least about 20 atoms, morepreferably at least about 50 atoms.

A wide variety of linkers are known in the art, which may be used toattach the nucleic acid probe to the solid support. The linker mayinclude an oligomer or polymer of any moiety or combination of moieties,which does not significantly interfere with the hybridization of thetarget sequence to the probe attached to the solid support. The linkermay include a nucleic acid, for example, a homopolymeric nucleic acid,which can be readily added on to the linker by automated synthesis.Alternatively, polymers such as polyethylene glycol can be used as thelinker. Such polymers are presently preferred over homopolymeric nucleicacids because they do not significantly interfere with the hybridizationof probe to the target nucleic acid. Polyethylene glycol is particularlypreferred because it is commercially available, soluble in both organicand aqueous media, easy to functionalize, and completely stable undernucleic acid synthesis and post-synthesis conditions.

The linkages between the solid support, the linker and the probe arepreferably not cleaved during synthesis or removal of base protectinggroups under basic conditions at high temperature. These linkages can,however, be selected from groups that are cleavable under a variety ofconditions. Examples of presently preferred linkages include carbamate,ester and amide linkages.

Peptide Probes

Peptides, proteins and peptide nucleic acids that are labeled with aquencher and a xanthene dye of the invention can be used in both in vivoand in vitro enzymatic assays.

Peptide constructs useful in practicing the invention include those withthe following features: i) a quencher; ii) a xanthene dye of theinvention; and iii) a cleavage or assembly recognition site for theenzyme. Moreover, the peptide construct is preferably exists in at leastone conformation that allows donor-acceptor energy transfer between thexanthene dye of the invention and the quencher when the fluorophore isexcited.

In the dual labeled probes of the invention, the donor and acceptormoieties are connected through an intervening linker moiety. The linkermoiety, preferably, includes a peptide moiety, but can be or can includeanother organic molecular moiety, as well. In a preferred embodiment,the linker moiety includes a cleavage recognition site specific for anenzyme or other cleavage agent of interest. A cleavage site in thelinker moiety is useful because when a tandem construct is mixed withthe cleavage agent, the linker is a substrate for cleavage by thecleavage agent. Rupture of the linker moiety results in separation ofthe xanthene dye and the quencher. The separation is measurable as achange in donor-acceptor energy transfer. Alternatively, peptideassembly can be detected by an increase in donor-acceptor energytransfer between a peptide fragment bearing a xanthene dye of theinvention and a peptide fragment bearing a donor moiety.

When the cleavage agent of interest is a protease, the linker generallyincludes a peptide containing a cleavage recognition sequence for theprotease. A cleavage recognition sequence for a protease is a specificamino acid sequence recognized by the protease during proteolyticcleavage. Many protease cleavage sites are known in the art, and theseand other cleavage sites can be included in the linker moiety. See,e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth.Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994);Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol.244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); Bouvier etal. Meth. Enzymol. 248: 614 (1995), Hardy et al., in AMYLOID PROTEINPRECURSOR IN DEVELOPMENT, AGING, AND ALZHEIMER'S DISEASE, ed. Masters etal. pp. 190-198 (1994).

Solid Support Immobilized Xanthene Dye Analogues

The xanthene dyes of the invention can be immobilized on substantiallyany polymer, biomolecule, or solid or semi-solid material having anyuseful configuration. Moreover, any conjugate comprising one or morexanthene dye of the invention can be similarly immobilized, through amoiety on either the xanthene or its conjugation partner. When thesupport is a solid or semi-solid, exemplary supports for immobilizationof the nucleic acid probe include controlled pore glass, glass plates,polystyrene, avidin coated polystyrene beads, cellulose, nylon,acrylamide gel and activated dextran. These solid supports arechemically stable, easily functionalized and have a well-defined surfacearea.

Solid supports such as, controlled pore glass (CPG, 500 Å, 1000 Å) andnon-swelling high cross-linked polystyrene (1000 Å) are exemplarypreferred supports.

According to the present invention, the surface of a solid support isfunctionalized with a xanthene dye of the invention or a species, e.g.,a linker or conjugation partner, to which a xanthene dye of theinvention is conjugated. For clarity of illustration, the followingdiscussion focuses on attaching a reactive xanthene dye of the inventionto a solid support. The following discussion is also broadly relevant toattaching to a solid support a species that includes within itsstructure a xanthene dye of the invention.

The xanthene dyes of the invention are preferably attached to a solidsupport by forming a bond between a reactive group on the xanthene dyeof the invention and a reactive group on the surface of the solidsupport, thereby derivatizing the solid support with one or morexanthene dyes of the invention. Alternatively, the reactive group on thexanthene dye of the invention is coupled with a reactive group on alinker arm attached to the solid support. The bond between the solidsupport and the xanthene dye of the invention is preferably a covalentbond, although ionic, dative and other such bonds are useful as well.Reactive groups which can be used in practicing the present inventionare discussed in detail above and include, for example, amines, hydroxylgroups, carboxylic acids, carboxylic acid derivatives, alkenes,sulfhydryls, siloxanes, etc.

A large number of solid supports appropriate for practicing the presentinvention are available commercially and include, for example, peptidesynthesis resins, both with and without attached amino acids and/orpeptides (e.g., alkoxybenzyl alcohol resin, aminomethyl resin,aminopolystyrene resin, benzhydrylamine resin, etc. (Bachem)),functionalized controlled pore glass (BioSearch Technologies, Inc.), ionexchange media (Aldrich), functionalized membranes (e.g., —COOHmembranes; Asahi Chemical Co., Asahi Glass Co., and Tokuyama Soda Co.),and the like.

Moreover, for applications in which an appropriate solid support is notcommercially available, a wide variety of reaction types are availablefor the functionalization of a solid support surface. For example,supports constructed of a plastic such as polypropylene, can be surfacederivatized by chromic acid oxidation, and subsequently converted tohydroxylated or aminomethylated surfaces. The functionalized support isthen reacted with a xanthene dye of the invention of complementaryreactivity, such as a xanthene dye of the invention active ester, acidchloride or sulfonate ester, for example. Supports made from highlycrosslinked divinylbenzene can be surface derivatized bychloromethylation and subsequent functional group manipulation.Additionally, functionalized substrates can be made from etched, reducedpolytetrafluoroethylene.

When the support is constructed of a siliceous material, such as glass,the surface can be derivatized by reacting the surface Si—OH, SiO—H,and/or Si—Si groups with a functionalizing reagent.

In a preferred embodiment, wherein the substrates are made from glass,the covalent bonding of the reactive group to the glass surface isachieved by conversion of groups on the substrate's surface by asilicon-modifying reagent such as:

(R^(a)O)₃—Si—R^(b)—X^(a)

where R^(a) is an alkyl group, such as methyl or ethyl, R^(b) is alinking group between silicon and X^(a), and X^(a) is a reactive groupor a protected reactive group. Silane derivatives having halogens orother leaving groups beside the displayed alkoxy groups are also usefulin the present invention. Exemplary linking groups include those thatinclude substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl groups.

In another preferred embodiment, the reagent used to functionalize thesolid support provides for more than one reactive group per each reagentmolecule. Using reagents, such as the compound below, each reactive siteon the substrate surface is, in essence, “amplified” to two or morefunctional groups:

(R^(a)O)₃—Si—Rb—(X^(a))_(n)

where R^(a) is an alkyl group (e.g., methyl, ethyl), R^(b) is a linkinggroup between silicon and X^(a), X^(a) is a reactive group or aprotected reactive group and n is an integer between 2 and 50, and morepreferably between 2 and 20. The amplification of a xanthene dye of theinvention by its attachment to a silicon-containing substrate isintended to be exemplary of the general concept of amplification. Thisamplification strategy is equally applicable to other aspects of theinvention in which a xanthene dye of the invention is attached toanother molecule or solid support.

A number of siloxane functionalizing reagents can be used, for example:

1. Hydroxyalkyl siloxanes (Silylate surface, functionalize withdiborane, and H₂O₂ to oxidize to the alcohol)

a. allyl trichlorosilane→→3-hydroxypropyl

b. 7-oct-1-enyl trichlorchlorosilane→→8-hydroxyoctyl;

2. Diol (dihydroxyalkyl) siloxanes (silylate surface and hydrolyze todiol)

a. (glycidyl trimethoxysilane→(2,3-dihydroxypropyloxy)propyl;

3. Aminoalkyl siloxanes (amines requiring no intermediatefunctionalizing step);

a. 3-aminopropyl trimethoxysilane→aminopropyl Dimeric secondaryaminoalkyl siloxanes;

b. bis(3-trimethoxysilylpropyl)amine→bis(silyloxylpropyl)amine.

It will be apparent to those of skill in the art that an array ofsimilarly useful functionalizing chemistries is available when supportcomponents other than siloxanes are used. Thus, for example alkyl thiols(e.g., self-assembled monolayers), functionalized as discussed above inthe context of siloxane-modifying reagents, can be attached to metalfilms and subsequently reacted with a xanthene dye of the invention toproduce the immobilized compound of the invention.

Exemplary groups of use for R^(b) in the above described embodiments ofthe present invention include, but are not limited to, substituted orunsubstituted alkyl (e.g., substituted or unsubstituted arylalkyl,alkylamino, alkoxy), substituted or unsubstituted aryl (e.g.,substituted or unsubstituted arylalkyl, aryloxy and aryloxyalkyl), acyl(e.g., acylamino, acyloxy), mercapto, saturated or unsaturated cyclichydrocarbyl, substituted or unsubstituted heteroaryl (e.g., substitutedor unsubstituted heteroarylalkyl), substituted or unsubstitutedheterocycloalkyl, and combinations thereof.

Acrylamide-Immobilized Probes

In another exemplary embodiment, a species conjugated to a xanthene dyeof the invention is immobilized within a matrix, such as an acrylamidematrix. In a preferred embodiment, the immobilization is accomplished inconjunction with the “acrydite” process (see, Rehman et al., NucleicAcids Research, 27: 649-655 (1999)). The acrydite method allowsimmobilization of alkene labeled probes within a polymerizedpolyacrylamide network. When target mixes are run past the immobilizedprobe band under electrophoresis conditions, the target nucleic acid iscaptured substantially quantitatively. However, detection of this eventcurrently requires a second probe. In one embodiment, probes bearing axanthene dye of the invention, and/or a fluorophore, are immobilized inan acrylamide matrix and subsequently contacted with the target mix. Byusing fluorescent probes as capture probes, signals from target mixescan be directly detected in real time.

Microarrays

The present invention also provides microarrays including immobilizedxanthene dye of the invention and compounds (e.g., peptides, nucleicacids, bioactive agents, etc.) functionalized with xanthene dye of theinvention. Moreover, the invention provides methods of interrogatingmicroarrays using probes that are functionalized with xanthene dye ofthe invention. The immobilized species and the probes are selected fromsubstantially any type of molecule, including, but not limited to, smallmolecules, peptides, enzymes, nucleic acids and the like.

Nucleic acid microarrays consisting of a multitude of immobilizednucleic acids are revolutionary tools for the generation of genomicinformation, see, Debouck et al., in supplement to Nature Genetics,21:48-50 (1999). The discussion that follows focuses on the use of axanthene dye of the invention in conjunction with nucleic acidmicroarrays. This focus is intended to be illustrative and does notlimit the scope of materials with which this aspect of the presentinvention can be practiced.

In another preferred embodiment, the compounds of the present inventionare utilized in a microarray format. The xanthene dye of the invention,or species bearing xanthene dye of the invention can themselves becomponents of a microarray or, alternatively they can be utilized as atool to screen components of a microarray.

Thus, in a preferred embodiment, the present invention provides a methodof screening a microarray. The method includes contacting the members ofthe microarray with, for example, a xanthene dye of theinvention-bearing probe and interrogating the microarray for regions offluorescence. In an exemplary embodiment, fluorescent regions areindicative of the presence of an interaction between the xanthene dye ofthe invention-bearing probe and a microarray component.

In another exemplary embodiment, the array comprises an immobilizedxanthene-bearing donor-acceptor energy transfer probe. In thisembodiment, when the probe interacts (e.g., hybridizes) with its target,energy transfer between the xanthene and a quencher moiety is disruptedand the xanthene dye fluoresces. Such arrays are easily prepared andread, and can be designed to give quantitative data. Arrays comprising axanthene-bearing probe are valuable tools for expression analysis andclinical genomic screening.

In another embodiment, the immobilized xanthene-bearing probe is not adonor-acceptor energy transfer probe. A microarray based on such asformat can be used to probe for the presence of interactions between ananalyte and the immobilized probe by, for example, observing thealteration of analyte fluorescence upon interaction between the probeand analyte.

Exemplary microarrays comprise n regions of identical or differentspecies (e.g., nucleic acid sequences, bioactive agents). In a preferredembodiment, n is a number from 2 to 100, more preferably, from 10 to1,000, and more preferably from 100 to 10,000. In a still furtherpreferred embodiment, the n regions are patterned on a substrate as ndistinct locations in a manner that allows the identity of each of the nlocations to be ascertained.

In yet another preferred embodiment, the invention also provides amethod for preparing a microarray of n xanthene-bearing probes. Themethod includes attaching xanthene dye-bearing probes to selectedregions of a substrate. A variety of methods are currently available formaking arrays of biological macromolecules, such as arrays of nucleicacid molecules. The following discussion focuses on the assembly of amicroarray of xanthene-bearing probes, this focus is for reasons ofbrevity and is intended to be illustrative and not limiting.

One method for making ordered arrays of xanthene-bearing probes on asubstrate is a “dot blot” approach. In this method, a vacuum manifoldtransfers a plurality of aqueous samples of probes, e.g., 96, from wellsto a substrate. The probe is immobilized on the porous membrane bybaking the membrane or exposing it to UV radiation. A common variant ofthis procedure is a “slot-blot” method in which the wells have highlyelongated oval shapes.

Another technique employed for making ordered arrays of probes uses anarray of pins dipped into the wells, e.g., the 96 wells of a microtiterplate, for transferring an array of samples to a substrate, such as aporous membrane. One array includes pins that are designed to spot amembrane in a staggered fashion, for creating an array of 9216 spots ina 22×22 cm area. See, Lehrach, et al., HYBRIDIZATION FINGERPRINTING INGENOME MAPPING AND SEQUENCING, GENOME ANALYSIS, Vol. 1, Davies et al,Eds., Cold Springs Harbor Press, pp. 39-81 (1990).

An alternate method of creating ordered arrays of probes is analogous tothat described by Pirrung et al. (U.S. Pat. No. 5,143,854, issued 1992),and also by Fodor et al., (Science, 251: 767-773 (1991)). This methodinvolves synthesizing different probes at different discrete regions ofa particle or other substrate. This method is preferably used withrelatively short probe molecules, e.g., less than 20 bases. A relatedmethod was described by Southern et al. (Genomics, 13: 1008-1017(1992)).

Khrapko, et al., DNA Sequence, 1: 375-388 (1991) describes a method ofmaking an nucleic acid matrix by spotting DNA onto a thin layer ofpolyacrylamide. The spotting is done manually with a micropipette.

The substrate can also be patterned using techniques such asphotolithography (Kleinfield et al., J. Neurosci. 8: 4098-120 (1998)),photoetching, chemical etching and microcontact printing (Kumar et al.,Langmuir 10: 1498-511 (1994)). Other techniques for forming patterns ona substrate will be readily apparent to those of skill in the art.

The size and complexity of the pattern on the substrate is limited onlyby the resolution of the technique utilized and the purpose for whichthe pattern is intended. For example, using microcontact printing,features as small as 200 nm are layered onto a substrate. See, Xia, Y.,J. Am. Chem. Soc. 117: 3274-75 (1995). Similarly, usingphotolithography, patterns with features as small as 1 μm are produced.See, Hickman et al., J. Vac. Sci. Technol. 12: 607-16 (1994). Patternswhich are useful in the present invention include those which includefeatures such as wells, enclosures, partitions, recesses, inlets,outlets, channels, troughs, diffraction gratings and the like.

In a presently preferred embodiment, the patterning is used to produce asubstrate having a plurality of adjacent wells, indentations or holes tocontain the probes. In general, each of these substrate features isisolated from the other wells by a raised wall or partition and thewells do not readily fluidically communicate. Thus, a particle, reagentor other substance, placed in a particular well remains substantiallyconfined to that well. In another preferred embodiment, the patterningallows the creation of channels through the device whereby an analyte orother substance can enter and/or exit the device.

In another embodiment, the probes are immobilized by “printing” themdirectly onto a substrate or, alternatively, a “lift off” technique canbe utilized. In the lift off technique, a patterned resist is laid ontothe substrate, and a probe is laid down in those areas not covered bythe resist and the resist is subsequently removed. Resists appropriatefor use with the substrates of the present invention are known to thoseof skill in the art. See, for example, Kleinfield et al., J. Neurosci.8: 4098-120 (1998). Following removal of the photoresist, a secondprobe, having a structure different from the first probe can be bondedto the substrate on those areas initially covered by the resist. Usingthis technique, substrates with patterns of probes having differentcharacteristics can be produced. Similar substrate configurations areaccessible through microprinting a layer with the desiredcharacteristics directly onto the substrate. See, Mrkish et al. Ann.Rev. Biophys. Biomol. Struct. 25:55-78 (1996).

Linkers

As used herein, the term “linker,” refers to a constituent of aconjugate between a xanthene dye and a carrier molecule. The linker is acomponent of the xanthene dye, the carrier molecule or it is a reactivecross-linking species that reacts with both the carrier molecule and thexanthene dye. The linker groups can be hydrophilic (e.g., tetraethyleneglycol, hexaethylene glycol, polyethylene glycol) or they can behydrophobic (e.g., hexane, decane, etc.). Exemplary linkers includesubstituted or unsubstituted C₆-C₃₀ alkyl groups, polyols (e.g.,glycerol), polyethers (e.g., poly(ethyleneglycol)), polyamines, aminoacids (e.g., polyaminoacids), saccharides (e.g., polysaccharides) andcombinations thereof.

In an exemplary embodiment, the linker joins donor and/or acceptormoieties and other groups to a nucleic acid, peptide or other componentof a probe. In a further exemplary embodiment, using a solid support,the immobilized construct includes a linker attached through the solidsupport and also to the xanthene dye.

In certain embodiments, it is advantageous to have the donor and/oracceptor moieties of the probe attached to a carrier molecule by a groupthat provides flexibility and distances the linked species from thecarrier molecule. Using linker groups, the properties of the donorand/or acceptor moiety is modulated. Properties that are usefullycontrolled include, for example, hydrophobicity, hydrophilicity,surface-activity, the distance of the quencher and/or xanthene dye ofthe invention moiety from the other probe components (e.g., carriermolecule) and the distance of the quencher from the xanthene dye of theinvention.

In an exemplary embodiment, the linker serves to distance the xanthenedye of the invention from a nucleic acid to which it is attached.Linkers with this characteristic have several uses. For example, axanthene dye of the invention held too closely to the nucleic acid maynot interact with the quencher group, or it may interact with too low ofan affinity. When a xanthene dye of the invention is itself stericallydemanding, the interaction leading to quenching can be undesirablyweakened, or it may not occur at all, due to a sterically inducedhindering of the approach of the two components.

When the construct comprising the xanthene dye is immobilized byattachment to, for example, a solid support, the construct can alsoinclude a linker moiety placed between the reactive group of the solidsupport and the xanthene analogue, or other probe component bound to thesolid support.

In yet a further embodiment, a linker group used in the probes of theinvention is provided with a group that can be cleaved to release abound moiety, e.g., a xanthene dye of the invention, quencher, minorgroove binder, intercalating moiety, and the like from the polymericcomponent. Many cleaveable groups are known in the art. See, forexample, Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshiet al., J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J.Immunol., 124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155:141-147 (1986); Park et al., J. Biol. Chem., 261: 205-210 (1986);Browning et al., J. Immunol., 143: 1859-1867 (1989). Moreover a broadrange of cleavable, bifunctional (both homo- and hetero-bifunctional)linker arms is commercially available from suppliers such as Pierce.Exemplary cleaveable groups are those cleaved by light, e.g.,nitrobenzyl derivatives, phenacyl groups, benzoin esters; hydrolysis,e.g., esters, carbonates; changes in pH, etc.

The Methods

In another aspect of the embodiment, the present invention provides amethod for detecting a target species in an assay mixture or othersample. The following discussion is generally relevant to the assaysdescribed herein. This discussion is intended to illustrate theinvention by reference to certain preferred embodiments and should notbe interpreted as limiting the scope of probes and assay types in whichthe compounds of the invention find use. Other assay formats utilizingthe compounds of the invention will be apparent to those of skill in theart.

An exemplary method uses a xanthene dye of the invention or a conjugatethereof to detect a nucleic acid target sequence. The method includes:(a) contacting the target sequence with a detector nucleic acid thatincludes a xanthene dye of the invention and a quencher; (b) hybridizingthe detector nucleic acid to the target sequence, thereby altering theconformation of the detector nucleic acid, causing a change in afluorescence parameter; and (c) detecting the change in the fluorescenceparameter, thereby detecting the nucleic acid target sequence.

In the methods described herein, unless otherwise noted, a preferreddetector nucleic acid includes a single-stranded target bindingsequence. The binding sequence has linked thereto: i) a quencher; andii) a xanthene dye of the invention. Moreover, prior to itshybridization to a complementary sequence, the detector nucleic acid ispreferably in a conformation that allows donor-acceptor energy transferbetween the quencher and the xanthene dye of the invention when thefluorophore is excited. Furthermore, in the methods described in thissection, a change in fluorescence is detected as an indication of thepresence of the target sequence. The change in fluorescence ispreferably detected in real time.

Presently preferred nucleic acid probes do not require the carriermolecule to adopt a secondary structure for the probe to function.Exemplary probes according to this motif include a quencher moiety thatincludes the diazo-linked quenchers described in co-pending, commonlyassigned U.S. patent application Ser. No. 09/567,863 or theconformationally assisted probes disclosed in U.S. patent applicationSer. No. 09/591,185.

In other methods described in this section, the detector nucleic acidcan assume substantially any intramolecularly associated secondarystructure, e.g., hairpins, stem-loop structures, pseudoknots, triplehelices and conformationally assisted structures. Moreover, theintramolecularly base-paired secondary structure preferably comprises aportion of the target binding sequence.

In another aspect, the invention provides a method for detectingamplification of a target sequence. The method includes the use of anamplification reaction including the following steps: (a) hybridizingthe target sequence and a detector nucleic acid that includes a xanthenedye of the invention. The detector nucleic acid preferably includes asingle-stranded target binding sequence and an intramolecularlyassociated secondary structure 5′ to the target binding sequence. Atleast a portion of the detector sequence forms a single stranded tailwhich is available for hybridization to the target sequence; (b)extending the hybridized detector nucleic acid on the target sequencewith a polymerase to produce a detector nucleic acid extension productand separating the detector nucleic acid extension product from thetarget sequence; (c) hybridizing a primer to the detector nucleic acidextension product and extending the primer with the polymerase, therebylinearizing the intramolecularly associated secondary structure andproducing a change in a fluorescence parameter; and (d) detecting thechange in the fluorescence parameter, thereby detecting the targetsequence.

In yet a further aspect, the invention provides a method of ascertainingwhether a first nucleic acid and a second nucleic acid hybridize. Inthis method, the first nucleic acid includes a xanthene dye of theinvention. The method includes: (a) contacting the first nucleic acidwith the second nucleic acid; (b) detecting an alteration in afluorescent property of a member selected from the first nucleic acid,the second nucleic acid and a combination thereof, thereby ascertainingwhether the hybridization occurs.

In general, to determine the concentration of a target molecule, e.g., anucleic acid, it is preferable to first obtain reference data in whichconstant amounts of probe are contacted with varying amounts of target.The fluorescence emission of each of the reference mixtures is used toderive a graph or table in which target concentration is compared tofluorescence emission. For example, a probe that hybridizes to a nucleicacid ligand and has a stem-loop architecture with the 5′ and 3′ terminibeing the sites of quencher and xanthene labeling, can be used to obtainsuch reference data. The value of the fluorescence emission is thencompared with the reference data to obtain the concentration of thetarget in the test mixture.

The xanthene dyes and their conjugates described herein can be used insubstantially any nucleic acid probe format now known or laterdiscovered. For example, the xanthene dyes of the invention can beincorporated into probe motifs, such as Taqman™ probes (Held et al.,Genome Res. 6: 986-994 (1996), Holland et al., Proc. Nat. Acad. Sci. USA88: 7276-7280 (1991), Lee et al., Nucleic Acids Res. 21: 3761-3766(1993)), molecular beacons (Tyagi et al., Nature Biotechnology14:303-308 (1996), Jayasena et al., U.S. Pat. No. 5,989,823, issued Nov.23, 1999)) scorpion probes (Whitcomb et al., Nature Biotechnology 17:804-807 (1999)), sunrise probes (Nazarenko et al., Nucleic Acids Res.25: 2516-2521 (1997)), conformationally assisted probes (Cook, R.,copending and commonly assigned U.S. patent application Ser. No.09/591,185), peptide nucleic acid (PNA)-based light up probes (Kubistaet al., WO 97/45539, December 1997), double-strand specific DNA dyes(Higuchi et al, Bio/Technology 10: 413-417 (1992), Wittwer et al,BioTechniques 22: 130-138 (1997)) and the like. These and other probemotifs with which the present xanthene dyes can be used are reviewed inNONISOTOPIC DNA PROBE TECHNIQUES, Academic Press, Inc. 1992.

Peptides, proteins and peptide nucleic acids that are labeled with aquencher and a xanthene dye of the invention can be used in both in vivoand in vitro enzymatic assays.

Thus, in another aspect, the present invention provides a method fordetermining whether a sample contains an enzyme. The method comprises:(a) contacting the sample with a peptide construct that includes axanthene dye of the invention; (b) exciting the fluorophore; and (c)determining a fluorescence property of the sample, wherein the presenceof the enzyme in the sample results in a change in the fluorescenceproperty. In selected embodiments, a fluorescence property of the probeis measured prior to incubating the probe with the enzyme.

Peptide constructs useful in practicing the invention include those withthe following features: i) a quencher; ii) a xanthene dye of theinvention; and iii) a cleavage or assembly recognition site for theenzyme. Moreover, the peptide construct is preferably exists in at leastone conformation that allows donor-acceptor energy transfer between thexanthene dye of the invention and the quencher when the fluorophore isexcited.

When the probe is used to detect an enzyme, such as a degradative enzyme(e.g., protease), and a degree of donor-acceptor energy transfer that islower than an expected amount is observed, this is generally indicativeof the presence of an enzyme. The degree of donor-acceptor energytransfer in the sample can be determined, for example, as a function ofthe amount of fluorescence from the donor moiety, the amount offluorescence from the acceptor moiety, the ratio of the amount offluorescence from the donor moiety to the amount of fluorescence fromthe acceptor moiety or the excitation state lifetime of the donormoiety.

The assay also is useful for determining the amount of enzyme in asample. For example, by determining the degree of donor-acceptor energytransfer at a first and second time after contact between the enzyme andthe tandem construct, and determining the difference in the degree ofdonor-acceptor energy transfer. The difference in the degree ofdonor-acceptor energy transfer is related to the amount of enzyme in thesample, the activity of the enzyme towards the construct, or both.

The assay methods also can also be used to determine whether a compoundalters the activity of an enzyme, i.e., screening assays. Thus, in afurther aspect, the invention provides methods of determining the amountof activity of an enzyme in a sample from an organism. The methodincludes: (a) contacting a sample comprising the enzyme and the compoundwith a peptide construct that includes a xanthene dye of the invention;(b) exciting the fluorophore; and (c) determining a fluorescenceproperty of the sample, wherein the activity of the enzyme in the sampleresults in a change in the fluorescence property. Peptide constructsuseful in this aspect of the invention are substantially similar tothose described immediately above.

In a preferred embodiment, the amount of enzyme activity in the sampleis determined as a function of the degree of donor-acceptor energytransfer in the sample and the amount of activity in the sample iscompared with a standard activity for the same amount of the enzyme. Adifference between the amount of enzyme activity in the sample and thestandard activity indicates that the compound alters the activity of theenzyme.

Representative enzymes with which the present invention can be practicedinclude, for example, trypsin, enterokinase, HIV-1 protease, prohormoneconvertase, interleukin-1b-converting enzyme, adenovirus endopeptidase,cytomegalovirus assemblin, leishmanolysin, β-secretase for amyloidprecursor protein, thrombin, renin, angiotensin-converting enzyme,cathepsin-D and a kininogenase, and proteases in general.

Proteases play essential roles in many disease processes such asAlzheimer's, hypertension, inflammation, apoptosis, and AIDS. Compoundsthat block or enhance their activity have potential as therapeuticagents. Because the normal substrates of peptidases are linear peptidesand because established procedures exist for making non-peptidicanalogs, compounds that affect the activity of proteases are naturalsubjects of combinatorial chemistry. Screening compounds produced bycombinatorial chemistry requires convenient enzymatic assays.

Convenient assays for proteases are based on donor-acceptor energytransfer from a donor fluorophore to a quencher placed at opposite endsof a short peptide chain containing the potential cleavage site (see,Knight C. G., Methods in Enzymol. 248:18-34 (1995)). Proteolysisseparates the fluorophore and quencher, resulting in increased intensityin the emission of the donor fluorophore. Existing protease assays useshort peptide substrates incorporating unnatural chromophoric aminoacids, assembled by solid phase peptide synthesis.

Assays of the invention are also useful for determining andcharacterizing substrate cleavage sequences of proteases or foridentifying proteases, such as orphan proteases. In one embodiment themethod involves the replacement of a defined linker moiety amino acidsequence with one that contains a randomized selection of amino acids. Alibrary of fluorescent xanthene dye probes, wherein the xanthene dyes ofthe invention are linked by a randomized peptide linker moiety, whichcan be generated using recombinant engineering techniques or syntheticchemistry techniques. Screening the members of the library can beaccomplished by measuring a signal related to cleavage, such asdonor-acceptor energy transfer, after contacting the cleavage enzymewith each of the library members of the tandem fluorescent peptideconstruct. A degree of donor-acceptor energy transfer that is lower thanan expected amount indicates the presence of a linker sequence that iscleaved by the enzyme. The degree of donor-acceptor energy transfer inthe sample can be determined, for example, as a function of the amountof fluorescence from the donor moiety, the amount of fluorescence fromthe acceptor donor moiety, or the ratio of the amount of fluorescencefrom the donor moiety to the amount of fluorescence from the acceptormoiety or the excitation state lifetime of the donor moiety.

Multiplex Analyses

In another exemplary embodiment, the xanthene dyes of the invention areutilized as a component of one or more probes used in a multiplex assayfor detecting one or more species in a mixture.

Probes that include a xanthene dye are particularly useful in performingmultiplex-type analyses and assays. In a typical multiplex analysis, twoor more distinct species (or regions of one or more species) aredetected using two or more probes, wherein each of the probes is labeledwith a different fluorophore, quencher or fluorophore/quencher pair.Preferred species used in multiplex analyses relying on donor-acceptorenergy transfer meet at least two criteria: the fluorescent species isbright and spectrally well resolved; and the energy transfer between thefluorescent species and the quencher is efficient.

Thus, in a further embodiment, the invention provides a mixturecomprising at least a first carrier molecule and a second carriermolecule. The first carrier molecule has covalently bound thereto afirst quencher and a first xanthene dye of the invention. An exemplaryquencher has a structure that includes at least three radicals selectedfrom aryl, substituted aryl, heteroaryl, substituted heteroaryl andcombinations thereof. At least two of the radicals are covalently linkedvia an exocyclic diazo bond. The mixture also includes a second carriermolecule. The fluorophore, quencher or both the fluorophore and quencherattached to the second carrier molecule is different than that attachedto the first nucleic acid. Exemplary quenchers of use in conjunctionwith the compounds of the invention include those described in commonlyowned WO 01/86001.

The xanthene dye of the invention allows for the design of multiplexassays in which more than one quencher structure is used in the assay.In one exemplary assay, at least two distinct xanthene dyes of theinvention are used with a common quencher structure. The quencher(s) canbe bound to the same molecule as the xanthene dye of the invention or toa different molecule. Moreover, the carrier molecules of use in aparticular assay system can be the same or different.

In addition to those embodiment described above, the present inventionalso provides a method for detecting and/or quantifying a particularmolecular species. The method includes: (a) contacting the species witha mixture such as that described above; and (b) detecting a change in afluorescent property of one or more component of the mixture, themolecular species or a combination thereof, thereby detecting and/orquantifying the molecular species.

Because the present invention provides readily available, reactivexanthene dyes, which can be “tuned” to emit fluorescence of a desiredwavelength, the compounds of the invention are particularly well suitedfor use in multiplex applications. Access to xanthene dyes of theinvention having a range of emission characteristics allows for thedesign of donor-acceptor energy transfer probes in which the acceptorabsorbance properties and the emission properties of the xanthene aresubstantially matched, thereby providing a useful level of spectraloverlap. Moreover, the xanthene dyes of the invention provide access toprobes that emit light at different wavelengths, allowing the probes tobe spectrally resolved, which is desirable for multiplex analysis.

The simultaneous use of two or more probes using donor-acceptor energytransfer is known in the art. For example, multiplex assays usingnucleic acid probes with different sequence specificities have beendescribed. Fluorescent probes have been used to determine whether anindividual is homozygous wild type, homozygous mutant or heterozygousfor a particular mutation. For example, using one quenched-fluoresceinmolecular beacon that recognizes the wild-type sequence and anotherrhodamine-quenched molecular beacon that recognizes a mutant allele, itis possible to genotype individuals for the β-chemokine receptor(Kostrikis et al. Science 279:1228-1229 (1998)). The presence of only afluorescein signal indicates that the individual is wild type, and thepresence of rhodamine signal only indicates that the individual is ahomozygous mutant. The presence of both rhodamine and fluorescein signalis diagnostic of a heterozygote. Tyagi et al. Nature Biotechnology 16:49-53 (1998)) have described the simultaneous use of four differentlylabeled molecular beacons for allele discrimination, and Lee et al.,BioTechniques 27: 342-349 (1999) have described seven color homogenousdetection of six PCR products. The compounds of the invention are of usein such methods.

The dyes of the present invention can be used in multiplex assaysdesigned to detect and/or quantify substantially any species, including,for example, whole cells, viruses, proteins (e.g., enzymes, antibodies,receptors), glycoproteins, lipoproteins, subcellular particles,organisms (e.g., Salmonella), nucleic acids (e.g., DNA, RNA, andanalogues thereof), polysaccharides, lipopolysaccharides, lipids, fattyacids, non-biological polymers and small molecules (e.g., toxins, drugs,pesticides, metabolites, hormones, alkaloids, steroids).

Kits

In another aspect, the present invention provides kits containing one ormore xanthene dye of the invention or a conjugate thereof. In oneembodiment, a kit includes a reactive xanthene dye of the invention anddirections for attaching this derivative to another molecule. In anotherembodiment, the kit includes a xanthene-labeled carrier, e.g., a nucleicacid that optionally is also labeled with a quencher and directions forusing this nucleic acid in one or more assay format. Other formats forkits will be apparent to those of skill in the art and are within thescope of the present invention.

The materials and methods of the present invention are furtherillustrated by the examples that follow. These examples are offered toillustrate, but not to limit the claimed invention.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

EXAMPLES Example 1 1.1 Preparation of Rhodamine B Acid Chloride 1

To a 500 mL round bottom flask were added 80% rhodamine B (6.0 g, 5.0mmol) and phosphorus oxychloride (50 mL). The flask was fitted with acondenser and calcium sulfate drying tube. The reaction mixture washeated at reflux for 16 h and then cooled to room temperature. Thevolatile components were removed under high vacuum. Acetonitrile (100mL) was added to dissolve the residue and was then removed by rotaryevaporation and high vacuum. The solid material was again dissolved inacetonitrile (100 mL) and stripped to dryness to afford crude rhodamineB acid chloride.

1.2 Rhodamine B N-methylaminobutanol hexafluorophosphate 2

Crude rhodamine B acid chloride (5.13 g, approx. 8.2 mmol) was dissolvedin a mixture of DMF (20 mL) and acetonitrile (70 mL) and to thissolution was added a solution of N-methylaminobutanol (3.0 g, 29.1 mmol)and triethylamine (7 mL) in acetonitrile (10 mL). The flask was fittedwith a septum and was warmed at 60° C. overnight. The volatilecomponents were removed under high vacuum and the residue waspartitioned between dichloromethane (200 mL) and 1N aqueous hydrochloricacid (200 mL). The aqueous phase was washed with dichloromethane (3×200mL). The combined organic solutions were washed with aqueous potassiumhexafluorophosphate solution (3×100 mL of 1 g/100 ml), dried (MgSO₄)filtered and evaporated in vacuo to afford a red solid. The solid wasdissolved in pyridine and stripped to dryness under high vacuum at 90°C. overnight to afford crude rhodamine B, N-methylaminobutanolhexafluorophosphate as a red solid (4.11 g, 74%).

1.3 Rhodamine B N-methylaminobutanol phosphoramidite 3

Dry crude rhodamine B N-methylaminobutanol (2.24 g, 4.0 mmol) wasdissolved in dry dichloromethane (50 mL) and then 1-H-tetrazole (0.070g, 1.0 mmol) was added. N,N,N′,N′-tetraisopropyl β-cyanoethyl phosphane(1.5 g, 5 mmol) was dissolved in dry dichloromethane (10 mL) and thissolution was added to that of the dye. The flask was fitted with aseptum and the mixture was stirred at room temperature for 4 h. Thesolution was diluted with dichloromethane (120 mL) and then was washedwith saturated sodium bicarbonate solution (2×60 mL), dried (MgSO₄),filtered and evaporated in vacuo to leave an oil. The oil was washedwith diethyl ether (3×50 mL) and the resulting solid residue wasdissolved in pyridine (60 mL). The pyridine was removed under highvacuum and the residue was dissolved in dichloromethane (5 mL). Removalof the dichloromethane in vacuo afforded the phosphoramidite as a redsolid (2.9 g).

1.4 Rhodamine B N-methylaminoethanol chloride 4

To a 500 mL round bottom flask were added 80% rhodamine B (12.2 g, 20.3mmol) and phosphorus oxychloride (120 mL). The flask was fitted with acondenser and calcium sulfate drying tube. The reaction mixture washeated at reflux for 16 h and then cooled to room temperature. Thevolatile components were removed under high vacuum. Acetonitrile (100mL) was added to dissolve the residue and was then removed by rotaryevaporation and high vacuum. The solid material was again dissolved inacetonitrile (100 mL) and to this solution was added a solution ofN-methylaminoethanol (5.0 g, 66.6 mmol) and triethylamine (20 mL) inacetonitrile (100 mL). The mixture was stirred at room temperatureovernight and then the volatile components were removed in vacuo. Theresidue was partitioned between dichloromethane (300 mL) and 1N aqueoushydrochloric acid (200 mL). The aqueous phase was washed withdichloromethane (3×300 mL). The combined organic solutions were dried(MgSO₄) filtered and evaporated in vacuo to afford rhodamine BN-methylaminoethanol chloride as a red solid (12.9 g).

1.5 Rhodamine B N-methylaminoethanol hexafluorophosphate 5

Rhodamine B N-methylaminoethanol chloride (4.0 g, approx. 6 mmol) wasdissolved in dichloromethane (500 mL) and the solution was washed withaqueous potassium hexafluorophosphate solution (3×100 mL of 1 g/100 mL).The organic phase was dried (MgSO₄), filtered and evaporated in vacuo.The residue was dissolved in pyridine (100 mL) and resulting solutionwas evaporated under high vacuum to afford rhodamine B,N-methylaminoethanol hexafluorophosphate as a dry purple solid.

1.6 Rhodamine B N-methylaminoethanol phosphoramidite 6

Rhodamine B, N-methylaminoethanol hexafluorophosphate (2.45 g, approx.3.8 mmol) was dissolved in dichloromethane (50 mL) and then1-H-tetrazole (0.07 g, 1.0 mmol) was added.N,N,N′,N′-tetraisopropyl-β-cyanoethyl phosphane (1.67 g, 5.5 mmol) wasdissolved in dichloromethane (10 mL) and the solution was added to thatof the dye. The mixture was stirred at room temperature for 4 h. Thesolution was diluted with dichloromethane (120 mL) and then was washedwith saturated sodium bicarbonate solution (2×60 mL), dried (MgSO₄),filtered and evaporated in vacuo to leave an oil. The oil was washedwith diethyl ether (3×60 mL) and the resulting solid residue wasdissolved in pyridine (60 mL). The pyridine was removed under highvacuum at 45° C. and then the residue was dissolved in dichloromethane(5 mL). Removal of the dichloromethane in vacuo afforded thephosphoramidite as a red foam.

1.7 Rhodamine B N-methylaminoethanoxy hydroxyhexyl urethanehexafluorophosphate 7

To a 250 mL round bottom flask were added rhodamine B ethanolhexafluorophosphate (1.75 g, 2.71 mmol) and dry pyridine (30 mL). Themixture was stirred while a solution of p-nitrophenylchloroformate (0.73g, 3.62 mmol) in dioxane (15 mL) was added over 5 min. The flask wasfitted with a septum and stirring was continued for a further 2 h. Asecond solution of p-nitrophenylchloroformate (0.44 g, 2.18 mmol) indioxane (10 mL) was added and the mixture was stirred for 96 h. Thevolatile components were removed in vacuo and the residue was dissolvedin dichloromethane (100 mL). The solution was washed with water (60 mL),dried (MgSO₄), and filtered. After concentrating to approx. 30 mL, thesolution was added portion-wise over 5 min. to a solution of6-aminohexanol (3.3 g, 28.2 mmol, excess) and diisopropylethylamine (3mL) in dichloromethane (33 mL). The mixture was stirred for 90 minutesand then diluted to 250 mL with dichloromethane. The solution was washedwith 1N aqueous hydrochloric acid (2×50 mL), dried (MgSO₄), filtered andevaporated in vacuo to afford a red solid that was subjected to columnchromatography on silica gel (6×20 cm) using 5% methanol indichloromethane as eluant. The pure fractions were evaporated in vacuoand the residue was dissolved in dichloromethane (30 mL) and filteredthrough a medium frit. Evaporation of the solvent afforded the urethanederivative as a red solid (1.32 g, 62%).

1.8 Rhodamine B N-methylaminoethanoxy hydroxyhexyl urethanephosphoramidite 8

Rhodamine B N-methylaminoethanoxyhydroxyhexyl urethanehexafluorophosphate (0.825 g, 1.0 mmol) was dissolved in pyridine andthen stripped to dryness under high vacuum at 80° C. overnight. It wasdissolved in dry dichloromethane (30 mL) and then 1-H-tetrazole (0.050g, 0.71 mmol) was added. N,N,N′,N′-tetraisopropyl-β-cyanoethyl phosphane(0.50 g, 1.7 mmol) was dissolved in dry dichloromethane (10 mL) and thissolution was added to that of the dye. The flask was fitted with aseptum and the mixture was stirred at room temperature for 3 h. Thesolution was diluted with dichloromethane (120 mL) and then was washedwith saturated sodium bicarbonate solution (2×60 mL), dried (MgSO₄),filtered and evaporated in vacuo to leave an oil. The oil was washedwith diethyl ether (3×60 mL) and the resulting solid residue wasdissolved in pyridine (60 mL). The pyridine was removed under highvacuum at 45° C. and the residue was dissolved in dichloromethane (5mL). Removal of the dichloromethane in vacuo afforded thephosphoramidite as a red foam (0.91 g, 88%).

Example 2 2.1 Preparation of Rhodamine 6G Acid 9

Rhodamine 6G (17.3 g, 36.4 mmol) was dissolved in DMSO (320 mL) and 1Naqueous sodium hydroxide (80 mL) was added. The mixture was stirred for16 h and then was neutralized by addition of 1N aqueous hydrochloricacid. The solid was filtered off and was dissolved in the minimum amountof methanol (approx. 500 mL). The solution was added to 1N aqueoushydrochloric acid (1200 mL) and then the methanol was boiled off. Aftercooling the solid was filtered off and was washed with water (2×30 mL).The solid was dried to afford the R6G acid (16 g, 97%).

2.2 Preparation of Rhodamine R6G succinimidyl ester 10

To a 100 mL round bottom flask was added rhodamine R6G acid (6.63 g,14.7 mmol), TSTU (5.25 g, 17.4 mmol), DMF (150 mL) anddiisopropylethylamine (9 mL). The mixture was stirred at roomtemperature for 20 h. The solid was filtered off, was washed with DMF(2×4 mL) and acetonitrile (2×5 mL), and was dried to afford rhodamineR6G succinimidyl ester (2.9 g, 36%).

2.3 Rhodamine R6G N-methylaminobutanol 11

To a 50 mL flask were added rhodamine R6G succinimidyl ester (1.0 g,1.83 mmol), N-methylaminobutanol (1.16 g, 11.3 mmol) and DMF (5 mL). Themixture was heated at 60° C. for 18 hours and then was diluted withwater (150 mL). The solution was applied to a column of C-18 reversedphase silica gel (3×13 cm). The column was eluted with a sharp gradientof 0 to 100% methanol to remove the impurities and then with acidifiedmethanol (5 mL of 1N aq. HCl in 1 L methanol) to elute the product.Evaporation of the eluant in vacuo afforded rhodamine R6GN-methylaminobutanol (0.81 g, 83%).

2.4 Preparation of Rhodamine 6 G N-methylaminoethanol 12

Commercially available Rhodamine 6 G (50 g) was heated to 120° C. withN-methylethanolamine (150 mL). The reaction mixture was checked after 1h by MALDI mass spectroscopy showing clean conversion into product, M/e474. The hot solution was poured carefully into 2N HCl (1200 mL) andchilled overnight. A red solid was collected by filtration and washedwith 0.5 N HCl (300 mL). The material was air dried for several days,then subjected to high vacuum for 18 h to give Rhodamine 6 GN-methylaminoethanol (38 g).

2.5 Preparation of 13, p-nitrophenylcarbonate of 12

Rhodamine 6 G N-methylaminoethanol (38 g) was dissolved in dry pyridine(800 mL) and a solution of p-nitrophenyl chloroformate (30 g) in drydioxane (400 mL) was added dropwise over 30 min. After 1 h of additionalstirring, a mass spectrum of an aliquot revealed conversion to acompound of M/e 641, consistent with the p-nitrophenyl carbonate ester.The solution was stripped to a tar by rotary evaporation andre-dissolved in 1 L of dichloromethane. This solution was washed withwater, 800 mL and then stripped to an oil by rotary evaporation.

2.6 Preparation of Rhodamine 6 G N-methylaminoethanoxy hydroxyhexylurethane 14

A solution of 40 g 6-amino-1-hexanol in 700 mL THF and 500 mL saturatedaq. Na₂CO₃ was prepared, and 13 was dissolved in 500 mL THF and addedover 20 min. The bright red mixture was stirred for an additional hr,whereupon a mass spectrum revealed conversion to a compound of M/e 618,consistent with the urethane derivative. Most of the THF was removed byrotary evaporation, and the aqueous residue was extracted withdichloromethane (800 mL). The dichloromethane layer was washed withwater (500 mL) followed by brine (500 mL). The organic phase was reducedto about 300 mL by rotary evaporation and applied to a column packedwith a (10×40 cm) bed of basic alumina in 2% methanol indichloromethane. The column was eluted at 100 mL/min with this samemobile phase until early running colored bands eluted, then the methanolwas increased to 4%. The product eluted as a bright orange band:fractions collected from this band were checked by TLC (20% methanol, 2%pyridine in dichloromethane). Pure fractions (r_(f)=0.6) were pooled andevaporated to give 14 (15 g).

2.7 Preparation of Rhodamine 6 G N-methylaminoethanoxy hydroxyhexylurethane phosphoramidite 15

Compound 14 (15 g) was dried by rotary evaporation from dry pyridine(400 mL) and high vacuum for 24 h. The compound was then dissolved indry dichloromethane (150 mL) and a premixed solution ofN,N,N′,N′-tetraisopropyl betacyanoethyl phosphane (7.5 g) and tetrazole(500 mg) in dry acetonitrile (150 mL) were added. After 2 h, TLC (sameconditions as above) showed conversion to a new compound (r_(f)=0.7),and a mass spectrum of an aliquot showed M/e 820, consistent withformation of the phosphoramidite. The solution was reduced by rotaryevaporation to a tar, then re-dissolved in dichloromethane (600 mL). Thesolution was washed with 5% aqueous Na₂CO₃ solution (400 mL) and driedover MgSO₄. Evaporation, followed by chromatography and evaporation asabove gave pure 15 as a red foam (19 g).

Example 3 3.1 Preparation of 19 from Rhodamine 101

Compound 19 and it precursors 16-18 were prepared in a manner analogousto corresponding compounds of Example 2.

Example 4 4.1 Preparation of Dichlorophenyl Xanthene Dye

4-Chlororesorcinol (33 g) was mixed with 4,5-dichlorophthalic anhydridein methane sulfonic acid (100 mL) according to the procedure of Menchen,et. al, (U.S. Pat. No. 5,654,442). Briefly, with magnetic stirring, thesolution was heated to 180° C. over 30 min and maintained for anadditional 30 minutes. The mixture was allowed to cool to ˜100° C. andwas cautiously poured into water (1 L). The solid was collected byfiltration and air dried for several days, followed by 24 h of highvacuum and was characterized as having the structure above.

4.2 Preparation of the Dichlorophenyl Ethyl Ester 20

The compound from Example 4.1 (50 g) was refluxed with absolute ethanol(1 L), which contained chlorotrimethylsilane (60 mL). After 2 h thesolution was cooled and the volatile components were removed by rotaryevaporation. High vacuum was applied to the orange solid for 24 h togive 20, (55 g).

4.3 Preparation of N-methanolaminoethanol amide 21

Ethyl ester 20 (7 g) was mixed with N-methylethanolamine (30 mL) andheated to 120° C., with magnetic stirring. After 30 min, the solutionwas poured cautiously into 2N HCl (1 L). The solid was collected byfiltration and air dried for several days, followed by 24 h of highvacuum to give 21 as a red solid (8 g).

4.4 Preparation of dimethoxytrityl ether 22 of N-methanolaminoethanolamide 21

Amino alcohol 21 (8 g) was dissolved in dry pyridine (200 mL) andDMT-chloride (10 g) was added. After stirring overnight, a mass spectrumof an aliquot showed a peak at 833 M/e, consistent with the addition ofthe DMT group to the aliphatic hydroxyl. The solvent was removed byrotary evaporation, and the residue was dissolved in dichloromethane(300 mL). The orange solution was washed with saturated NaHCO₃ (200 mL),followed by brine (200 mL). The crude material was then dissolved in asolution (50 mL) of 1% methanol, 1% TEA in dichloromethane. This wasapplied to a column of neutral alumina (5×20 cm), 7% by weight water,and eluted with 1 L of the above-described mobile phase. A gradient to4% methanol was then run over 4 liters of solvent. Pure fractionscontaining 22 (TLC rf 0.6, silica plates with 5% methanol, 1% TEA indichloromethane) were pooled and evaporated to give pure 22 (4 g).

4.5 Preparation of O-trimethyl acetyl phenyl derivative 23

22 (4 g) was dissolved in dry pyridine (300 mL) under argon. Trimethylacetyl chloride (5 mL) was then added dropwise via syringe over 3 min.The solution changed from bright orange to a dull yellow color duringthe addition. TLC (same conditions as above) showed a change to a yellowspot rf=0.8. A mass spectrum of an aliquot showed a peak at M/e 920,consistent with acylation of the phenolic type hydroxyl group. Themixture was purified with the same methods as the starting material togive 23 (2.4 g).

4.6 Removal of the DMT Group to Form 24

Compound 23 (2 g) was treated with a solution (300 mL) of 3%dichloroacetic acid in dichloromethane for 1 h. The acid was thenneutralized by cautiously adding saturated NaHCO₃, (300 mL) withstirring for an additional 1 h. The layers were separated and thedichloromethane layer was washed with water (200 mL) followed by brine(200 mL). The organic phase was dried over MgSO₄ and reduced by rotaryevaporation. The product was purified by column chromatography as aboveto give pure 24 (0.8 g).

4.7 Preparation of Phosphoramidite 25

Compound 24 was dried by rotary evaporation from dry pyridine (50 mL)and high vacuum overnight and reacted with a pre-mixed solution ofN,N,N′,N′-tetra-isopropyl-2-cyanoethyl phosphoramidate (500 mg) andtetrazole (20 mg) in dry acetonitrile (50 mL). After 1 h, a massspectrum of an aliquot showed a peak corresponding to conversion to anew compound, M/e 822, consistent with the formation of thephosphoramidite. The solution was reduced by rotary evaporation andre-dissolved ethyl acetate (200 mL). The organic phase was washed withsaturated NaHCO₃ (100 mL) and dried over MgSO₄. Filtration followed byevaporation and high vacuum overnight gave phosphoramidite 25 (1 g).

Example 5 5.1 Preparation of Rhodamine 6GN-methylaminoethanol-linker-nucleoside 26

Rhodamine 6G N-methylaminoethanol 13 (10 g) was dried by rotaryevaporation from dry pyridine (200 mL) and high vacuum for 24 h. Theresulting material was dissolved in dry pyridine (200 mL) and then drydioxane (75 mL) was added. p-Nitrophenyl chloroformate (12 g) was addeddropwise as a solution in dioxane (100 mL) over 20 min. The solution wasstirred for several hours whereupon a mass spectrum of an aliquotrevealed complete conversion to the p-nitrophenylcarbonate ester. Thesolvents were removed by rotary evaporation and the residue wasdissolved in dichloromethane (400 mL). The organic phase was washed withof 0.5 N KH₂PO₄ (2×300 mL) and was then dried over MgSO₄. The solutionwas reduced to an oil by rotary evaporation and re-dissolved in THF (150mL).N⁴-(2-(4,7,10-trioxa-1,13-tridecanediamine)-5-methyl-5′-(4,4′-dimethoxytrityl)-3′-O-tert-butyldimethylsilyl-2′deoxycytidine(Lyttle, et. al., Bioconjugate Chem. 13: 1146-1154 (2002)) (15 g) wasdissolved in THF (300 mL) and saturated NaHCO₃ (200 mL) was added. Withmagnetic stirring, the THF solution of p-nitrophenylcarbonate ester wasadded slowly over 5 min. The solution was stirred for 18 h. A massspectrum of an aliquot showed a peak at M/e 1363, consistent with thatof the desired product. Most of the THF was removed and the residue wasextracted with dichloromethane (300 mL). The organic phase was washedwith water (2×200 mL) and dried over MgSO₄. The solution was reduced byrotary evaporation and applied to a basic alumina column, 7% by weightwater (20×50 cm), packed with 2% methanol, 2% pyridine indichloromethane. The column was eluted with 2 L of this solvent, then agradient to 10% methanol was run over 20 L of solvent. Fractions werechecked by TLC (20% methanol, 2% pyridine in dichloromethane) and thosepure fractions having rf 0.6 were pooled and evaporated to give 26 (6 g)as a red tar.

5.2 Deprotection of the 3′-hydroxyl moiety, forming 27

Compound 26 (6 g) was dissolved in a solution of THF (100 mL), 1 Ntetrabutylammonium fluoride (12 mL) in THF (Aldrich #216143) and aceticacid (2 mL). The solution was allowed to stand overnight and an aliquot,checked by mass spectrum, showed a peak at M/e 1252, consistent withremoval of the TBDMS group. The reaction mixture was quenched by addingNaHCO₃ (20 mL). THF was removed by rotary evaporation. The residue wasdissolved in dichloromethane (300 mL). The organic phase was washed withwater (200 mL) followed by NaHCO₃ (200 mL). The dichloromethane layerwas dried over MgSO₄ and reduced to an oil by rotary evaporation. TLC(20% methanol, 2% pyridine in dichloromethane) revealed adequate productpurity to proceed without purification. The material was dried by rotaryevaporation with dry pyridine (100 mL) followed by high vacuum for 18 hto give 27 (2.5 g).

5.3 Preparation of 3′-glycolic acid ester 28

Compound 27 (2.5 g) was dissolved in dry pyridine (100 mL) andN-methylimidazole (0.5 mL) and diglycolic anhydride (2.5 g) were added.The mixture was allowed to stand 48 h, whereupon a mass spectrum of analiquot showed a peak at M/e 1367, consistent with esterification of thehydroxyl group of 27. The solvent was removed by rotary evaporation, andthe residue was dissolved in dichloromethane (300 mL). The organic phasewas washed with 0.5 M KH₂PO₄ (3×200 mL) and dried over MgSO₄. Thesolution was filtered and reduced by rotary evaporation to give 28 (1.5g) as a foam.

5.4 Conjugation of 28 to CPG, Forming Functionalized CPG 29

Controlled pore glass (CPG) for solid phase oligonucleotide synthesiswas made by activating 28 (300 mg) with BOP (250 mg) and N-methylmorpholine (50 μL) in acetonitrile (50 mL), and then adding 500 Åaminopropyl CPG (Biosearch) (5 g). The slurry was allowed to standovernight and the support was then rinsed thoroughly with acetonitrile.Unreacted aminopropyl groups were then capped with a mixture ofN-methylimidazole (5 mL) and acetic anhydride (5 mL) in acetonitrile (50mL). After 15 min the solution was rinsed out of the support and thedark orange CPG was washed thoroughly with acetonitrile, followed bydichloromethane and then was dried by high vacuum overnight. The finalloading, according to mild acid removal of the DMT groups, was 25micromoles per gram.

Example 6 6.1 Synthesis of Rhodamine B 4-hydroxypiperidinylN,N,diisopropyl betacyanoethyl phosphoramidite

Rhodamine B (37 g), was dissolved in DMF (400 mL) and N-methylmorpholine(20 mL) was added. BOP (45 g) was added as a solid, and the solution wasmagnetically stirred for 10 min. A solution of 4-hydroxypiperidine (20g) in DMF (200 mL) was added to the dye solution dropwise over 20 min.After an additional 30 min, a mass spectrum of an aliquot showedcomplete reaction (to the 4-hydroxy-piperidinyl alcohol, M/e 531). TheDMF was removed by rotary evaporation, and the residue was dissolved in10% MeOH in DCM (600 mL). The organic solution was washed with 1 N HCl(800 mL) and evaporated to a tar. Column chromatography on alumina witha gradient of 2-4% MeOH in DCM gave the product (29 g), rf 0.5 (silicaplate, 10% MeOH/DCM 2% pyridine). One half of the product was dried bypyridine strip and under vacuum overnight. A solution ofN,N,N,N-tetraisopropyl betacyanoethyl phosphane (6.6 g) and tetrazole(500 mg) in acetonitrile (200 mL) was added to a solution of the productin DCM (200 mL). After 2 h, a mass spectrum of an aliquot showedcomplete reaction (to the amidite, M/e 730). The organics were removedby rotary evaporation, and dissolved in DCM (500 mL). The organic phasewas washed with sat'd NaHCO₃ (300 mL) of evaporated to a tar. Theproduct was purified by chromatography on alumina with a gradient of2-4% MeOH in DCM, 1% pyridine 1% water. Fractions were pooled andevaporated to give amidite (14.6 g), rf 0.7 (silica plate, 10% MeOH/DCM2% pyridine).

6.2 Synthesis of Rhodamine 101 4-hydroxypiperidinyl N,N,diisopropylbetacyanoethyl phosphoramidite

Rhodamine 101 (55 g) was dissolved in DMF (800 mL) and ofN-methylmorpholine (25 mL) was added. BOP (50 g) was added as a solid,and the solution was magnetically stirred for 10 min. A solution of4-hydroxypiperidine (30 g) in DMF (200 mL) was prepared and this wasadded to the dye solution dropwise over 20 min. After and additional 60min. a mass spectrum of an aliquot showed complete reaction (to the4-hydroxypiperidinyl alcohol, M/e 577). The DMF was removed by rotaryevaporation, and the residue was dissolved in 10% MeOH in DCM (900 mL).The organic solution was washed with 1 N HCl (800 mL) and evaporated toa tar. Column chromatography on silica with a gradient of 2-6% MeOH inDCM, 2% pyridine, gave the intermediate alcohol (40.9 g), rf 0.5 (silicaplate, 20% MeOH/DCM 2% pyridine). One half of this product was dried bypyridine strip and vacuum overnight. A solution ofN,N,N,N-tetraisopropyl betacyanoethyl phosphane (10 g) and tetrazole(600 mg) was mixed in acetonitrile (300 mL) and added to a solution ofthe alcohol above dissolved in DCM (300 mL). After 4 h, a mass spectrumof an aliquot showed complete reaction (to the amidite, M/e 777). Thesolution was stripped by rotary evaporation, and re-dissolved in DCM(700 mL). The organic phase was washed with sat'd NaHCO₃ (500 mL) andevaporated to a tar. The product was purified by chromatography onalumina with a gradient of 2-4% MeOH in DCM, 2% pyridine 1% water.Fractions were pooled and evaporated to give amidite (17.3 g), rf 0.8(silica plate, 20% MeOH/DCM 2% pyridine).

Example 7 7.1 Synthesis of Nucleic Acids 5′ Labeled with 15, 19 and 25

DNA Fragments 3′-TTTTTTTTTT-5′ and 3′-TTCGATAAGTCTAG-5′ were made at a200 nm scale on 3′-glycolate CPGs (van der Laan, et. al, TetrahedronLett. 38: 2252 (1997)) with cyanoethyl phosphoramidite monomers on aBiosearch 8750™ DNA synthesizer. Protecting groups on the exocyclicamine groups of A, C and G were benzoyl, acetyl and dimethylformamidine,respectively. After the synthesis was complete, the 5′ DMT group wasremoved (with 3% dichloroacetic acid in dichloromethane) and thesynthesis column with the CPG containing the DNA was washed with dryacetonitrile.

To couple the dye moiety to the nucleic acid, 35-50 mg of 15, 19 and 25dye phosphoramidites were dissolved in dry acetonitrile (150 μL) in a 20mL scintillation vial, and activated molecular sieves (20 mg) wereadded. The resulting solution was applied to the column with a 1 mLsyringe. A companion syringe containing 100 μL of 0.4 MS-ethylthiotetrazole in acetonitrile was attached at the other end ofthe column. The solutions were mixed over the CPG with the syringes, andallowed to stand for 5 min. The columns were put back on the DNAsynthesizer and washed with acetonitrile followed by oxidizer solution(0.02 M iodine in a mixture of THF (70%):pyridine (20%):water (10%).After 30 seconds, this solution was washed with acetonitrile and thecontents of each column were expelled into 1.5 mL screw-cap Eppendorf™tubes. A mixture of 2-methoxyethylamine (Aldrich) (100 μL) and methanol(300 μL) were introduced into each tube, and the tubes were capped andallowed to stand for 6 h at room temperature. The methanol containingthe labeled DNA was removed, and the CPG was washed with fresh methanol(400 μL). The CPG was discarded and the methanol containing the labeledDNA fragments was evaporated.

7.2 Synthesis of Nucleic Acids 5′ Labeled with 6

DNA Fragments 3′-TTTTT-5′, 3′-TTTTTTTTTT-5′, DNA Fragments3′-TTTTTTTTTTTTTTT-5′, labeled with 6 were prepared as described above,except that the fragments were cleaved from the CPG support using asolution of t-butylamine (25%):methanol (25%):water (50%).

7.3 Synthesis of Nucleic Acids 3′ Labeled with 28

For 3′-dye labeled DNA synthesis CPG 27 was used in lieu of the standardDNA synthesis support. After automated DNA synthesis was finished, thesample was deprotected and cleaved with the amine/alcohol solution andevaporated after 6 h. The samples were dissolved in de-ionized water (1mL) for analysis.

7.4 HPLC and Mass Spectral Analysis of 3′ and 5′-Dye Labeled DNA

Anion exchange HPLC analyses were performed as follows: 2-20 μL of theaqueous samples, depending on the concentration, were injected onto aDionex anion exchange column (4.6×250 mm); samples were eluted at 2mL/min with aqueous buffers of (A) 0.025 M TRIS HCl and 0.01 M TRIS, and(B) 0.025 M TRIS HCl, 0.01 M TRIS, and 1.0 M NaBr using a lineargradient of 1:0 to 0:1 over 14 min, with UV detection at 260 nm. Reversephase HPLC as follows: 20 μL of the aqueous sample were injected onto aHAISIL HL C18, 5μ column (4.6×150 mm); samples were eluted at 1 mL/minwith buffers of (A) 0.1M TEAA, 5% acetonitrile, (B) acetonitrile, with alinear gradient of 1:0 to 0:1 over 15 min: UV detection at 260 nm.Samples for mass spectral analysis were prepared as per Bruker Corp.

7.5 Results

Table 2 shows calculated and found masses of the above Dye-DNA 15 mersby MALDI mass spectroscopy:

TABLE 2 Compound Calculated M/e Found 5′-25 15 mer 5160.2 5129 5′-15 15mer 5249.2 5214 5′-19 15 mer 5324.2 5304 3′-28 15 mer 5273.2 5241

Example 8 8.1 Evaluation of Dual Labeled Nucleic Acid Probes

The dual labeled probes were evaluated for the efficacy in a real-timePCR procedure. The real-time PCR procedure is performed on a real-timequantitative PCR device such as the ABI Prizm 7700 Sequence DetectionSystem™ (Applied Biosystems, Foster City, Calif.), or the iCycler(BioRad, Hercules, Calif.). These two systems amplify samples in a96-well format on a thermocycler. During amplification, light-inducedfluorescent signal is collected in real-time for all 96 wells, anddetected. The systems include software for running the instruments andfor analyzing the data.

The ABI 7700 Sequence Detection System was calibrated with xanthene dye15 and other fluorescent dyes.

Quantitation was obtained using primers and a dual-labeled probe derivedfrom sequence encoding the ApoB (apolipoprotein B) gene and from theTelomeraseRT (Telomerase reverse transcriptase) gene. BHQ1 and BHQ2quenchers, described in copending U.S. patent application Ser. No.09/567,863, were incorporated into the primers (see, for example, Waltonet al., Bioconjugate Chemistry 13: 1155-1158 (2002)). The xanthenes 15,19 and 25 were utilized to incorporate a fluorophore at the 5′-terminus.Gene-specific primers and fluorogenic probes were designed based uponthe coding sequences of the DNAs. The sequences for the primers andprobes (forward primer, reverse primer and probe) used for the ApoB andTelomerase are as follows:

TelomeraseRT.f1 CAGGTGGAGACCCTGAGAA TelomeraseRT.r1ACACCTTTGGTCACTCCAAAT TelomeraseRT.p1 TCCCAGAGCTCCCAGGGTCC ApoB.f1TGAAGGTGGAGGACATTCCTCTA ApoB.r1 CTGGAATTGCGATTTCTGGTAA ApoB.p1CGAGAATCACCCTGCCAGACTTCCGT

The ApoB probe sequence and the Telomerase probe sequence weresynthesized with various combinations of fluorescent dyes and quencher.These probes were used along with their gene specific primers inreal-time PCR assays. Human DNA (Clontech, Palo Alto, Calif.) wasdetected at concentrations of 100 ng per reaction or 1 ng per reactionin an assay. Data were analyzed from triplicate reactions, and theaverage and standard deviation for each triplicate was calculated.

Real-time quantitative PCR (Livak et al., PCR Methods Appl. 4(6): 357-62(1995)) was used to determine whether the compounds above comparedfavorably to existing dyes used as reporters in such an assay. Positiveresults in this assay can be interpreted to extend to positive resultsin other assays, including but not limited to, the Invader (Hall et al.,Natl. Acad. Sci. USA 97: 8272-8277 (2000)), the Amplifuor (Uehara,Biotechniques 26(3): 552-8 (1999)), the Scorpion (Thelwell et al., Nucl.Acids. Res. 28: 3752-3761 (2000)), and the Molecular Beacon (Tyagi &Kramer, Nature Biotechnol. 14: 303-308 (1996)).

The real-time PCR assay reaction is a fluorescent PCR-based techniquethat makes use of the 5′ exonuclease activity of Taq DNA polymeraseenzyme to monitor amplification in real time. Two oligonucleotideprimers are used to generate a PCR product typical of a PCR reaction. Athird oligonucleotide, or probe, is designed to detect nucleotidesequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areporter fluorescent dye and a fluorescence quencher, traditionallyTAMRA or DABCYL. Any light emission from the reporter dye is quenched bythe quenching dye when the two dyes are located close together as theyare on the intact probe. During the amplification reaction, the Taq DNApolymerase enzyme cleaves the probe in a template-dependent manner. Theresultant probe fragments disassociate in solution, and signal from thereleased reporter dye is free from the quenching effect of the quenchermoiety. One molecule of reporter dye is liberated for each new moleculesynthesized, and detection of the unquenched reporter dye provides thebasis for quantitative interpretation of the data.

The real-time PCR assay data are initially expressed as Ct, or thethreshold cycle. This is defined as the cycle at which the reportersignal accumulates above the background level of fluorescence. One unitcorresponds 1 PCR cycle or approximately a 2-fold amplification relativeto normal, two units corresponds to 4-fold, 3 units to 8-foldamplification, and so on. The Ct values are used as quantitativemeasurement of the relative number of starting copies of a particulartarget sequence in a nucleic acid sample.

The compounds of the invention are useful replacements for dyes that arecommonly used in nucleic acid assays. For example, compounds such as 15are a useful replacement for the fluorescent dyes JOE and HEX. The dyesROX and Texas Red can be replaced with compounds such as 19. Compoundssuch as 3, 6 and 8 can be substituted for TAMRA.

Excitation and emission wavelengths of the dyes of the invention and theart-recognized dyes are listed in Table 3. Because the Ct value for thedyes of the invention is equivalent to, or lower than, the Ct value ofthe art-recognized dyes, the present dyes are useful replacements forthe art recognized dyes.

TABLE 3 Excitation Emission Dye Maximum/nm Maximum/nm FAM 495 520 JOE520 548 HEX 535 556 VIC 538 554 TAMRA 555 576 ROX 575 602 25 522 544 15540 561 3, 6, 8 565 588 19 593 613

The results from three typical experiments were graphed and the data arepresented in FIG. 14, FIG. 15 and FIG. 16.

The present invention provides xanthenes that are functionalized with anoxygen-containing reactive functional group that can be used tofacilitate the conjugation of the xanthenes to a conjugation partner.The resulting fluorescent conjugate is of use in essentially any assayin which detection of a fluorescent species has a role. While specificexamples have been provided, the above description is illustrative andnot restrictive. Any one or more of the features of the previouslydescribed embodiments can be combined in any manner with one or morefeatures of any other embodiments in the present invention. Furthermore,many variations of the invention will become apparent to those skilledin the art upon review of the specification. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to theappended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

1. A xanthene dye having the formula:

in which R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are independentlyselected from substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocycloalkyl, halogen, H, NO₂, CN and C(Z¹)R¹⁴, NR¹⁵R¹⁶ and Z²R¹⁶;R³ is selected from Z²R¹⁶ and NR¹⁵R¹⁶ wherein Z¹ is a member selectedfrom O, S and NH; Z² is a member selected from O and S; R¹⁵ is a memberselected from H, substituted or unsubstituted alkyl, and substituted orunsubstituted heteroalkyl; R¹⁶ is selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl, C(Z³)R¹⁷,and a nitrogen-containing reactive group comprising R¹⁵ and R¹⁶,together with the nitrogen to which they are attached, wherein saidreactive group is a member selected from —NHNH₂, —N═C═S and —N═C═Owherein Z³ is a member selected from O, S and NH; R¹⁷ is a memberselected from substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, OR¹⁸, and NR¹⁹R²⁰ wherein R¹⁸ is a memberselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl and C(O)R²¹ wherein  R²¹ issubstituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl; R¹⁹ and R²⁰ are members independently selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl Y is a member selected from C(O) and S(O)₂; X is a memberselected from (NR²²R²³) and (O) wherein R²² and R²³ are membersindependently selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl; and R¹² and R¹³ are membersindependently selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl, with the proviso that at least one of R¹² orR¹³ comprises a member selected from a bond to a carrier molecule, abond to a linker bound to a carrier molecule, a bond to a solid support,a bond to a linker attached to a solid support, a bond to a fluorescencequencher, a bond to a linker to a fluorescence quencher and anoxygen-containing reactive group, and further with the proviso that whenR¹² and R¹³, together with the nitrogen to which they are attached forma piperazine ring said oxygen-containing reactive group is aphosphoramidite and said bond to a carrier molecule is other than a bondto a peptide.